U.S. patent application number 15/512711 was filed with the patent office on 2017-10-26 for high-purity steviol glycosides.
The applicant listed for this patent is The Coca-Cola Company, PureCircle Sdn Bhd. Invention is credited to Daniel AURIOL, Aurelien BADIE, Cynthia BUNDERS, Cyrille JARRIN, Avetik MARKOSYAN, Indra PRAKASH, Robert TER HALLE.
Application Number | 20170303565 15/512711 |
Document ID | / |
Family ID | 55533681 |
Filed Date | 2017-10-26 |
United States Patent
Application |
20170303565 |
Kind Code |
A1 |
MARKOSYAN; Avetik ; et
al. |
October 26, 2017 |
HIGH-PURITY STEVIOL GLYCOSIDES
Abstract
Methods of preparing highly purified steviol glycosides,
particularly rebaudiosides A, D and M are described. The methods
include utilizing recombinant microorganisms for converting various
staring compositions to target steviol glycosides. In addition,
novel steviol glycosides reb D2, reb M2, and reb I are disclosed,
as are methods of preparing the same. The highly purified
rebaudiosides are useful as non-caloric sweetener in edible and
chewable compositions such as any beverages, confectioneries,
bakery products, cookies, and chewing gums.
Inventors: |
MARKOSYAN; Avetik; (Yerevan,
AM) ; PRAKASH; Indra; (Alpharetta, GA) ;
JARRIN; Cyrille; (Muret, FR) ; BADIE; Aurelien;
(Labege, FR) ; TER HALLE; Robert; (Baziege,
FR) ; BUNDERS; Cynthia; (Atlanta, GA) ;
AURIOL; Daniel; (Roques, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PureCircle Sdn Bhd
The Coca-Cola Company |
Kuala Lumpur
NW Atlanta |
GA |
MY
US |
|
|
Family ID: |
55533681 |
Appl. No.: |
15/512711 |
Filed: |
August 21, 2015 |
PCT Filed: |
August 21, 2015 |
PCT NO: |
PCT/US2015/046354 |
371 Date: |
March 20, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2015/045906 |
Aug 19, 2015 |
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15512711 |
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62061359 |
Oct 8, 2014 |
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62062288 |
Oct 10, 2014 |
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62064630 |
Oct 16, 2014 |
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62082446 |
Nov 20, 2014 |
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62097387 |
Dec 29, 2014 |
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62185964 |
Jun 29, 2015 |
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62118132 |
Feb 19, 2015 |
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62052544 |
Sep 19, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Y 302/01021 20130101;
A23L 2/60 20130101; C12P 19/18 20130101; A23V 2002/00 20130101;
Y02P 20/582 20151101; C12P 19/56 20130101; C07H 15/256 20130101;
A23L 27/36 20160801; C07H 1/06 20130101; C12Y 204/01 20130101 |
International
Class: |
A23L 2/60 20060101
A23L002/60; C12P 19/18 20060101 C12P019/18; C12P 19/56 20060101
C12P019/56; A23L 27/30 20060101 A23L027/30 |
Claims
1. A method for producing highly purified target steviol glycoside
composition, comprising the steps of: a. providing a starting
composition comprising at least one organic compound; b. providing
a recombinant microorganism containing steviol biosynthesis
enzymes, UDP-glycosyltransferases, and optionally UDP-glucose
recycling enzymes; c. contacting the recombinant microorganism with
a medium comprising the starting composition to produce a
composition comprising a target steviol glycoside; and d.
separating the target steviol glycoside from the medium to provide
a highly purified target steviol glycoside composition.
2. The method of claim 1, wherein the staring composition is
selected from the group consisting of poyols, carbohydrates and
combinations thereof.
3. The method of claim 1, wherein the microorganism is selected
from the group consisting of E. coli, Saccharomyces sp.,
Aspergillus sp., Pichia sp., Bacillus sp., and Yarrowia sp.
4. The method of claim 1, wherein the target steviol glycoside is
selected from the group consisting of stevioside, reb A, reb D, reb
D2, reb M, reb M2, reb I and combinations thereof.
5. The method of claim 1, wherein the target steviol glycoside is
separated from the medium using crystallization, separation by
membranes, centrifugation, extraction, chromatographic separation
or a combination of such methods.
6. The method of claim 1, wherein the highly purified target
steviol glycoside composition comprises the target steviol
glycoside in an amount greater than about 95% by weight on a dry
basis.
7. (canceled)
8. (canceled)
9. A highly purified target steviol glycoside composition prepared
according to the method of claim 1, comprising the target steviol
glycoside content in an amount greater than about 95% by weight on
a dry basis.
10. (canceled)
11. A highly purified target steviol glycoside composition prepared
according to the method of claim 1, wherein the target steviol
glycoside is polymorphic.
12. (canceled)
13. A consumable product comprising the highly purified target
steviol glycoside composition of claim 1, wherein the product is
selected from the group consisting of a food, a beverage, a
pharmaceutical composition, a tobacco product, a nutraceutical
composition, an oral hygiene composition, and a cosmetic
composition.
14-20. (canceled)
21. A method for preparing reb I comprising a. contacting a
starting composition comprising reb A with an enzyme capable of
transforming reb A to reb I, UDP-glucose, and optionally
UDP-glucose recycling enzymes to produce a composition comprising
reb I; and b. isolating a composition comprising reb I.
22. The method of claim 22, further comprising purifying the
composition comprising reb I to provide reb I having a purity
greater than about 95% by weight on an anhydrous basis.
23-26. (canceled)
27. A method for enhancing the sweetness of a beverage comprising a
sweetener comprising the steps of: a.) providing a beverage
comprising a sweetener; and b.) adding a sweetness enhancer
selected from reb D2, reb M2, reb I or a combination thereof;
wherein the sweetness enhancer is present in a concentration at or
below the sweetness recognition threshold of the sweetness
enhancer.
28. The method of claim 1, wherein the UDP-glycosyltransferase is
selected from the group consisting of UGT76G1 variants, having
greater than 75% amino-acid sequence identity with UGT76G1.
29. The method of claim 1, wherein the UDP-glycosyltransferase is
selected from the group consisting of UGTSL2 variants, having
greater than 75% amino-acid sequence identity with UGTSL2.
30. A method for making target steviol glycoside comprising
converting starting steviol glycoside to target steviol glycoside
using a UDP-glucosyltransferase.
31. The method of claim 30, wherein the UDP-glycosyltransferase is
selected from the group consisting of UGT76G1 variants, having
greater than 75% amino-acid sequence identity with UGT76G1.
32. The method of claim 30, wherein the UDP glycosyltransferase is
selected from the group consisting of UGTSL2 variants, having
greater than 75% amino-acid sequence identity with UGTSL2.
33. The method of claim 1, further comprising providing an enzyme
with .beta.-glucosidase activity for hydrolysis of reb D2 and or
reb M2.
Description
TECHNICAL FIELD
[0001] The present invention relates to a biocatalytic process for
preparing compositions comprising steviol glycosides, including
highly purified steviol glycoside compositions. The present
invention also relates to novel steviol glycosides, methods for
isolation of the same and uses for the novel steviol
glycosides.
BACKGROUND OF THE INVENTION
[0002] High intensity sweeteners possess a sweetness level that is
many times greater than the sweetness level of sucrose. They are
essentially non-caloric and are commonly used in diet and
reduced-calorie products, including foods and beverages. High
intensity sweeteners do not elicit a glycemic response, making them
suitable for use in products targeted to diabetics and others
interested in controlling for their intake of carbohydrates.
[0003] Steviol glycosides are a class of compounds found in the
leaves of Stevia rebaudiana Bertoni, a perennial shrub of the
Asteraceae (Compositae) family native to certain regions of South
America. They are characterized structurally by a single base,
steviol, differing by the presence of carbohydrate residues at
positions C13 and C19. They accumulate in Stevia leaves, composing
approximately 10%-20% of the total dry weight. On a dry weight
basis, the four major glycosides found in the leaves of Stevia
typically include stevioside (9.1%), rebaudioside A (3.8%),
rebaudioside C (0.6-1.0%) and dulcoside A (0.3%). Other known
steviol glycosides include rebaudioside B, C, D, E, F and M,
steviolbioside and rubusoside.
[0004] Although methods are known for preparing steviol glycosides
from Stevia rebaudiana, many of these methods are unsuitable for
use commercially.
[0005] Accordingly, there remains a need for simple, efficient, and
economical methods for preparing compositions comprising steviol
glycosides, including highly purified steviol glycoside
compositions.
[0006] Additionally, there remains a need for novel steviol
glycosides and methods of preparing and isolating the same.
SUMMARY OF THE INVENTION
[0007] The present invention provides a biocatalytic process for
preparing a composition comprising a target steviol glycoside by
contacting a starting composition comprising an organic substrate
with a microorganism and/or biocatalyst, thereby producing a
composition comprising a target steviol glycoside.
[0008] The starting composition comprises an organic compound. In
one embodiment, the starting composition is selected from the group
consisting of polyols and various carbohydrates.
[0009] The target steviol glycoside can be any steviol glycoside.
In one embodiment, the target steviol glycoside is steviolmonoside,
steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside
B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F,
rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H,
rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M,
rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N,
rebaudioside O or a synthetic steviol glycoside.
[0010] In one embodiment, the target steviol glycoside is
stevioside.
[0011] In another embodiment, the target steviol glycoside is
rebaudioside A.
[0012] In still another embodiment, the target steviol glycoside is
rebaudioside D.
[0013] In yet another embodiment, the target steviol glycoside is
rebaudioside M.
[0014] The microorganism can be any microorganism comprising at
least one biocatalyst suitable for converting the starting
composition to target steviol glycosides.
[0015] The biocatalysts can be located on the surface and/or inside
the microorganism.
[0016] The biocatalysts include the steviol biosynthesis enzymes
and UDP-glycosyltransferases (UGTs), or their variants, having
greater than 75% amino-acid sequence identity.
[0017] In one embodiment the steviol biosynthesis enzymes include
mevalonate (MVA) pathway enzymes.
[0018] In another embodiment the steviol biosynthesis enzymes
include non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway
(MEP/DOXP) enzymes.
[0019] In one embodiment the steviol biosynthesis enzymes are
selected from the group including geranylgeranyl diphosphate
synthase, copalyl diphosphate synthase, kaurene synthase, kaurene
oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthetase,
deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose
5-phosphate reductoisomerase (DXR),
4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS),
4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK),
4-diphosphocytidyl-2-C-methyl-D-erythritol 2,4-cyclodiphosphate
synthase (MCS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate
synthase (HDS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate
reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA
reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate
pyrophosphate decarboxylase, cytochrome P450 reductase etc.
[0020] The UDP-glucosyltransferase can be any
UDP-glucosyltransferase capable of adding at least one glucose unit
to the steviol and or steviol glycoside substrate to provide the
target steviol glycoside.
[0021] The microorganism may be any suitable microorganism. In one
embodiment, the microorganism may be, for example, E. coli,
Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp.,
Yarrowia sp. etc. In another embodiment, the
UDP-glucosyltransferases are synthesized.
[0022] In one embodiment, the UDP-glucosyltransferase is selected
from group including UGT74G1, UGT85C2, UGT76G1, UGT91D2 or their
variants, having greater than 75% amino-acid sequence identity.
[0023] In one embodiment, the UDP-glucosyltransferase is any
UDP-glucosyltransferase capable of adding at least one glucose unit
to rubusoside to form stevioside. In a particular embodiment, the
UDP-glucosyltransferase is UGT91D2 or UGT91D2 variant, having
greater than 75% amino-acid sequence identity with UGT91D2.
[0024] In one embodiment, the UDP-glucosyltransferase is any
UDP-glucosyltransferase capable of adding at least one glucose unit
to stevioside to form rebaudioside A. In a particular embodiment,
the UDP-glucosyltransferase is UGT76G1 or UGT76G1 variant, having
greater than 75% amino-acid sequence identity with UGT76G1.
[0025] In another embodiment, the UDP-glucosyltransferase is any
UDP-glucosyltransferase capable of adding at least one glucose unit
to rebaudioside A to form rebaudioside D. In a particular
embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT91D2
variant, having greater than 75% amino-acid sequence identity with
UGT91D2.
[0026] In yet another embodiment, the UDP-glucosyltransferase is
any UDP-glucosyltransferase capable of adding at least one glucose
unit to rebaudioside D to form rebaudioside M. In a particular
embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT76G1
variant, having greater than 75% amino-acid sequence identity with
UGT76G1.
[0027] In yet another embodiment, the UDP-glucosyltransferase is
any UDP-glucosyltransferase capable of adding at least one glucose
unit to rebaudioside I to form rebaudioside M. In a particular
embodiment, the UDP-glucosyltransferase is UGTSL or UGTSL variant,
having greater than 75% amino-acid sequence identity with
UGTSL.
[0028] In yet another embodiment, the UDP-glucosyltransferase is
any UDP-glucosyltransferase capable of adding at least two glucose
units to rebaudioside E to form rebaudioside M. In a particular
embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT76G1
variant, having greater than 75% amino-acid sequence identity with
UGT76G1.
[0029] Optionally, the method of the present invention further
comprises recycling UDP to provide UDP-glucose. In one embodiment,
the method comprises recycling UDP by providing a recycling
catalyst and a recycling substrate, such that the biotransformation
of the steviol glycoside substrate to the target steviol glycoside
is carried out using catalytic amounts of UDP-glucosyltransferase
and UDP-glucose (FIG. 3).
[0030] In one embodiment, the recycling catalyst is sucrose
synthase.
[0031] In one embodiment, the recycling substrate is sucrose.
[0032] Optionally, the method of the present invention further
comprises purifying the composition comprising the target steviol
glycoside. The composition comprising the target steviol glycoside
can be purified by any suitable method, such as, for example,
crystallization, separation by membranes, centrifugation,
extraction, chromatographic separation or a combination of such
methods.
[0033] In one embodiment, purification produces a composition
comprising greater than about 80% by weight of the target steviol
glycoside on an anhydrous basis. In another embodiment,
purification produces a composition comprising greater than about
90% by weight of the target steviol glycoside. In particular
embodiments, the composition comprises greater than about 95% by
weight of the target steviol glycoside.
[0034] The target steviol glycoside can be in any polymorphic or
amorphous form, including hydrates, solvates, anhydrous or
combinations thereof.
[0035] The present invention also provides consumable products
comprising compositions prepared by the disclosed methods. Suitable
consumer products include, but are not limited to, food, beverages,
pharmaceutical compositions, tobacco products, nutraceutical
compositions, oral hygiene compositions, and cosmetic
compositions.
[0036] The present invention also provides novel steviol glycosides
reb D2 and reb M2, which are isomers of reb D and reb M,
respectively. In one embodiment, isolated and purified reb D2 is
provided. In another embodiment, isolated and purified reb M2 is
provided. Reb D2 and reb M2 may also be present in any consumable
products disclosed herein. In a particular embodiment, beverages
comprising reb D2 and/or reb M2 are provided.
[0037] Methods of preparing reb D2 and reb M2 are also provided
herein. Both are formed during the biotransformation of reb A to
reb D. Reb M2 is believed to form from biotransformation of reb D2
in situ.
[0038] Methods of selective hydrolysis of 1,6-.beta.-glucosidic
linkages in reb D2 and/or reb M2, by enzyme with
.beta.-1,6-glucosidase activity, are also provided herein.
[0039] In one embodiment for selective hydrolysis of
1,6-.beta.-glucosidic linkages in reb D2 and/or reb M2, at least
one enzyme is selected from the group including, glycosidase
(NC-IUBMB EC 3.2.1), glucosidase, glucanase, Isolase (011410;
National Enzyme Company, USA), Aromase (GLY0151441; Amano Enzyme,
Japan), naringinase (NAH0550102; Amano Enzyme, Japan), cellulase
(e.g. Cellulase from Trichoderma reesei ATCC 26921; Sigma C2730),
cellobiase (e.g. Cellobiase from Aspergillus niger, Sigma C6105),
Viscozyme L (Sigma V2010), etc.
[0040] In one embodiment, the present invention is a method for the
preparation of a composition comprising reb D2 comprising: (a)
contacting a starting composition comprising reb A with an enzyme
capable of transforming reb A to reb D2, UDP-glucose, and
optionally UDP-glucose recycling enzymes, to produce a composition
comprising reb D2, and (b) isolating the composition comprising reb
D2.
[0041] In another embodiment, the present invention is a method for
the preparation of a composition comprising reb M comprising (a)
contacting a starting composition comprising reb D with an enzyme
capable of transforming reb D to reb M, UDP-glucose, and optionally
UDP-glucose recycling enzymes, to produce a composition comprising
reb M, and (b) and isolating the composition comprising reb M.
[0042] A further embodiment, the present invention is a method for
the preparation of a composition comprising reb M comprising (a)
contacting a starting composition comprising reb A with an enzyme
capable of transforming reb A to reb D, UDP-glucose, and optionally
UDP-glucose recycling enzymes, to produce a composition comprising
reb D, (b) optionally, isolating the composition comprising reb D,
(c) contacting the composition comprising reb D with an enzyme
capable of transforming reb D to reb A UDP-glucose, and optionally
UDP-glucose recycling enzymes to produce a composition comprising
reb M, and (d) isolating the composition comprising reb M.
[0043] The composition can be further purified to provide reb D or
reb M with purities greater than about 95% by weight on a dry
basis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] The accompanying drawings are included to provide a further
understanding of the invention. The drawings illustrate embodiments
of the invention and together with the description serve to explain
the principles of the embodiments of the invention.
[0045] FIG. 1 shows the structure of reb M.
[0046] FIG. 2 shows the biocatalytic production of reb M from
stevioside.
[0047] FIG. 3 shows the biocatalytic production of reb A from
stevioside using the enzyme UGT76G1 and concomitant recycling of
UDP to UDP glucose via sucrose synthase.
[0048] FIG. 4 shows the IR spectrum of reb M.
[0049] FIG. 5. shows the HPLC chromatogram of the product of the
biocatalytic production of reb M from reb D, as detailed in Example
14. The peak with retention time of 24.165 minutes corresponds to
unreacted reb D. The peak with retention time of 31.325 minutes
corresponds to reb M.
[0050] FIG. 6. shows the HPLC chromatogram of purified reb M
produced by biocatalysis from reb D.
[0051] FIG. 7 shows the HPLC chromatogram of a reb M standard.
[0052] FIG. 8 shows the HPLC chromatogram of co-injection of a reb
M standard and reb M purified from biotransformation from reb
D.
[0053] FIG. 9 shows an overlay of the .sup.1H NMR spectra of a reb
M standard and reb M purified following biosynthesis from reb
D.
[0054] FIG. 10 shows the HRMS spectrum of reb M purified following
biocatalytic production from reb D.
[0055] FIG. 11 shows LC-MS analysis of semi-synthetic steviol
glycoside mixture, Lot number CB-2977-106, showing TIC (A), MS of
peak at 1.8 min (B), MS of reb M2 peak at 4.1 min (C), MS of reb D
peak at 6.0 min (D), MS of reb D2 peak at 7.7 min (E), MS of peak
at 9.4 min (F), MS of rebaudioside Apeak at 15.2 min (G), MS of
peak at 16.5 min (H), and MS of peak at 18.3 min (I).
[0056] FIG. 12 shows the trace of semi-synthetic steviol glycoside
mixture, Lot number CB-2977-106. Chromatogram gridlines are not
homogeneous as the detector was re-calibrated 14 min following
injection.
[0057] FIG. 13 shows HPLC analysis of semi-synthetic steviol
glycoside mixture, Lot number CB-2977-106 (A), Isolated reb M2 (B),
isolated reb D (C) and isolated reb D2 (D).
[0058] FIG. 14 shows the .sup.1H NMR spectrum of reb D2 (500 MHz,
pyridine-d.sub.5).
[0059] FIG. 15 shows the .sup.13C NMR spectrum of reb D2(125 MHz,
pyridine-d.sub.5).
[0060] FIG. 16 shows an expansion of the .sup.13C NMR spectrum of
reb D2 (125 MHz, pyridine-d.sub.5).
[0061] FIG. 17 shows the .sup.1H-.sup.1H COSY Spectrum of reb D2
(500 MHz, pyridine-d.sub.5).
[0062] FIG. 18 shows the HSQC-DEPT spectrum of reb D2(500 MHz,
pyridine-d.sub.5).
[0063] FIG. 19 shows the HMBC spectrum of reb D2.
[0064] FIG. 20 shows an expansion of HMBC spectrum of reb D2 (500
MHz, pyridine-d.sub.5).
[0065] FIG. 21 shows the .sup.1H NMR spectrum of reb M2(500 MHz,
D.sub.2O).
[0066] FIG. 22 shows the .sup.13C NMR spectrum of reb M2 (125 MHz,
D.sub.2O/TSP).
[0067] FIG. 23 shows an expansion of the .sup.13C NMR spectrum of
reb M2 (125 MHz, D.sub.2O/TSP).
[0068] FIG. 24 shows the .sup.1H-.sup.1H COSY spectrum of reb M2
(500 MHz, D.sub.2O).
[0069] FIG. 25 shows the HSQC-DEPT spectrum of reb M2(500 MHz,
D.sub.2O).
[0070] FIG. 26 shows the HMBC spectrum of reb M2 (500 MHz,
D.sub.2O).
[0071] FIG. 27 shows an expansion of HMBC spectrum of reb M2 (500
MHz, D.sub.2O).
[0072] FIG. 28 shows an HPLC chromatogram for the analysis done in
Example 47.
[0073] FIG. 29 shows an HPLC chromatogram for the analysis done in
Example 47.
[0074] FIG. 30 shows an LC-CAD analysis done in Example 47.
[0075] FIG. 31 shows an ESI-TOF mass spectrogram as described in
Example 47.
[0076] FIG. 32 shows a mass spectrogram as described in Example
47.
[0077] FIG. 33 shows an MS/MS spectrogram as described in Example
47.
[0078] FIG. 34 shows an MS/MS spectrogram as described in Example
47.
[0079] FIG. 35 shows the results of .sup.1H NMR as described in
Example 47.
[0080] FIG. 36 shows the results of .sup.1H NMR as described in
Example 47.
[0081] FIG. 37 shows the results of .sup.1H NMR as described in
Example 47.
[0082] FIG. 38 shows the results of .sup.13C NMR as described in
Example 47.
[0083] FIG. 39 shows the results of .sup.13C NMR as described in
Example 47.
[0084] FIG. 40 shows the results of .sup.1H-.sup.1H COSY as
described in Example 47.
[0085] FIG. 41 shows the results of HSQC-DEPT as described in
Example 47.
[0086] FIG. 42 shows the results of HMBC as described in Example
47.
[0087] FIG. 43 shows the results of HMBC as described in Example
47.
[0088] FIG. 44 shows the results of NOESY as described Example
47.
[0089] FIG. 45 shows the results of NOESY as described Example
47.
[0090] FIG. 46 shows the results of 1D TOCSY as described in
Example 47.
[0091] FIG. 47 shows the results of 1D TOCSY as described in
Example 47.
[0092] FIG. 48 shows the results of 1D TOCSY as described in
Example 47.
[0093] FIG. 49 shows the results of 1D TOCSY as described in
Example 47.
[0094] FIG. 50 shows the results of 1D TOCSY as described in
Example 47.
[0095] FIG. 51 shows an HPLC (CAD) graph showing conversion of
stevioside to rebaudioside A.
[0096] FIG. 52 shows an HPLC (CAD) graph showing conversion of
rebaudioside D to rebaudioside M.
[0097] FIG. 53a-e show HPLC chromatograms showing HPLC assay
results for Example 20.
[0098] FIG. 54 shows an HPLC chromatogram showing the HPLC assay
results for Example 21.
[0099] FIG. 55a-e show HPLC chromatograms showing the HPLC assay
results for Example 22.
[0100] FIG. 56a-b show HPLC chromatograms showing the HPLC assay
results for Example 23.
[0101] FIG. 57a-b show LC-MS spectrograms showing the LC-MS assay
results for Example 24.
[0102] FIG. 58 shows a graph showing the reaction profile for
Example 25.
[0103] FIG. 59a-b show HPLC chromatograms showing the HPLC assay
results for Example 28.
[0104] FIG. 60a-b show HPLC chromatograms showing the HPLC assay
results for Example 29.
[0105] FIG. 61 shows an HPLC chromatogram showing the HPLC assay
results for Example 30.
[0106] FIG. 62 shows an LS-MS spectrogram showing the LS-MS assay
results for Example 31.
[0107] FIG. 63a-c show HPLC chromatograms showing the HPLC assay
results for Example 32.
[0108] FIG. 64 shows an HPLC chromatogram showing the HPLC assay
results for Example 35.
[0109] FIG. 65 shows an HPLC chromatogram showing the HPLC assay
results for Example 37
[0110] FIG. 66 shows a graph showing the HPLC results for Example
43.
[0111] FIG. 67 shows a graph showing the reaction profile for
Example 46.
[0112] FIG. 68a-f show reaction profiles for Example 49.
[0113] FIG. 69a-c show graphs showing the HPLC results for Example
50.
[0114] FIG. 70a-d show reaction profile graphs for Example 51.
[0115] FIG. 71 shows a reaction profile graph for Example 52.
[0116] FIG. 72a shows a reaction profile graph for Example 54.
[0117] FIG. 72b shows an HPLC chromatogram showing the HPLC
analysis for Example 54.
[0118] FIG. 73a shows a reaction profile graph for Example 55.
[0119] FIG. 73b shows an HPLC chromatogram showing the HPLC
analysis for Example 55.
[0120] FIG. 74a shows a reaction profile graph for Example 56.
[0121] FIG. 74b shows an HPLC chromatogram showing the HPLC
analysis for Example 56.
[0122] FIG. 75a shows a reaction profile graph for Example 57.
[0123] FIG. 75b shows an HPLC chromatogram showing the HPLC
analysis for Example 57.
[0124] FIG. 76a shows a reaction profile graph for Example 58.
[0125] FIG. 76b shows an HPLC chromatogram showing the HPLC
analysis for Example 58.
DETAILED DESCRIPTION
[0126] The present invention provides a biocatalytic process for
preparing a composition comprising a target steviol glycoside by
contacting a starting composition comprising an organic substrate
with a microorganism, thereby producing a composition comprising a
target steviol glycoside.
[0127] One object of the invention is to provide an efficient
biocatalytic method for preparing steviol glycosides, particularly
stevioside, reb E, reb A, reb D, reb D2, reb M, and reb M2 from
various starting compositions.
[0128] As used herein, "biocatalysis" or "biocatalytic" refers to
the use of natural or genetically engineered biocatalysts, such as
cells, protein enzymes, to perform single or multiple step chemical
transformations on organic compounds. Biocatalysis include
fermentation, biosynthesis and biotransformation processes. Both,
isolated enzyme and whole-cell biocatalysis methods are known in
the art. Biocatalyst protein enzymes can be naturally occurring or
recombinant proteins.
[0129] All sequences listed herein, including any nucleic acid or
amino acid sequences, include variants having >75%, >80%,
>90%, >95%, >96%, >97%, >98%, or >99% sequence
identity to the nucleic acid or amino acid sequences described
herein.
[0130] As used herein, the term "steviol glycoside(s)" refers to a
glycoside of steviol, including, but not limited to, naturally
occurring steviol glycosides, e.g. steviolmonoside, steviolbioside,
rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside
G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A,
rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L,
rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D,
rebaudioside M2, rebaudioside D2, rebaudioside N, rebaudioside 0,
synthetic steviol glycosides, e.g. enzymatically glucosylated
steviol glycosides and combinations thereof.
[0131] Chemical structures of steviol and its glycosides
##STR00001##
TABLE-US-00001 Compound R.sub.1 R.sub.2 Steviol H H Steviolmonoside
H .beta.--Glc Steviol monoglucosyl ester .beta.--Glc H Rubusoside
.beta.--Glc .beta.--Glc Steviolbioside H
.beta.--Glc--.beta.--Glc(2.fwdarw.1) Stevioside .beta.--Glc
.beta.--Glc--.beta.--Glc(2.fwdarw.1) Rebaudioside A .beta.--Glc
##STR00002## Rebaudioside D .beta.--Glc--.beta.--Glc(2.fwdarw.1)
##STR00003## Rebaudioside E .beta.--Glc--.beta.--Glc(2.fwdarw.1)
.beta.--Glc--.beta.--Glc(2.fwdarw.1) Rebaudioside M ##STR00004##
##STR00005## (Glc = glucose)
[0132] Starting Composition
[0133] As used herein, "starting composition" refers to any
composition (generally an aqueous solution) containing one or more
organic compound comprising at least one carbon atom.
[0134] In one embodiment, the starting composition is selected from
the group consisting of polyols and various carbohydrates.
[0135] The term "polyol" refers to a molecule that contains more
than one hydroxyl group. A polyol may be a diol, triol, or a
tetraol which contain 2, 3, and 4 hydroxyl groups, respectively. A
polyol also may contain more than four hydroxyl groups, such as a
pentaol, hexaol, heptaol, or the like, which contain 5, 6, or 7
hydroxyl groups, respectively. Additionally, a polyol also may be a
sugar alcohol, polyhydric alcohol, or polyalcohol which is a
reduced form of carbohydrate, wherein the carbonyl group (aldehyde
or ketone, reducing sugar) has been reduced to a primary or
secondary hydroxyl group. Examples of polyols include, but are not
limited to, erythritol, maltitol, mannitol, sorbitol, lactitol,
xylitol, inositol, isomalt, propylene glycol, glycerol, threitol,
galactitol, hydrogenated isomaltulose, reduced
isomalto-oligosaccharides, reduced xylo-oligosaccharides, reduced
gentio-oligosaccharides, reduced maltose syrup, reduced glucose
syrup, hydrogenated starch hydrolyzates, polyglycitols and sugar
alcohols or any other carbohydrates capable of being reduced.
[0136] The term "carbohydrate" refers to aldehyde or ketone
compounds substituted with multiple hydroxyl groups, of the general
formula (CH.sub.2O).sub.n, wherein n is 3-30, as well as their
oligomers and polymers. The carbohydrates of the present invention
can, in addition, be substituted or deoxygenated at one or more
positions. Carbohydrates, as used herein, encompass unmodified
carbohydrates, carbohydrate derivatives, substituted carbohydrates,
and modified carbohydrates. As used herein, the phrases
"carbohydrate derivatives", "substituted carbohydrate", and
"modified carbohydrates" are synonymous. Modified carbohydrate
means any carbohydrate wherein at least one atom has been added,
removed, or substituted, or combinations thereof. Thus,
carbohydrate derivatives or substituted carbohydrates include
substituted and unsubstituted monosaccharides, disaccharides,
oligosaccharides, and polysaccharides. The carbohydrate derivatives
or substituted carbohydrates optionally can be deoxygenated at any
corresponding C-position, and/or substituted with one or more
moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl,
acyloxy, amino, amido, carboxyl derivatives, alkylamino,
dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo,
mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl,
carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl,
phosphino, thioester, thioether, oximino, hydrazino, carbamyl,
phospho, phosphonato, or any other viable functional group provided
the carbohydrate derivative or substituted carbohydrate functions
to improve the sweet taste of the sweetener composition.
[0137] Examples of carbohydrates which may be used in accordance
with this invention include, but are not limited to, tagatose,
trehalose, galactose, rhamnose, various cyclodextrins, cyclic
oligosaccharides, various types of maltodextrins, dextran, sucrose,
glucose, ribulose, fructose, threose, arabinose, xylose, lyxose,
allose, altrose, mannose, idose, lactose, maltose, invert sugar,
isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose,
gulose, idose, talose, erythrulose, xylulose, psicose, turanose,
cellobiose, amylopectin, glucosamine, mannosamine, fucose,
glucuronic acid, gluconic acid, glucono-lactone, abequose,
galactosamine, beet oligosaccharides, isomalto-oligosaccharides
(isomaltose, isomaltotriose, panose and the like),
xylo-oligosaccharides (xylotriose, xylobiose and the like),
xylo-terminated oligosaccharides, gentio-oligosaccharides
(gentiobiose, gentiotriose, gentiotetraose and the like), sorbose,
nigero-oligosaccharides, palatinose oligosaccharides,
fructooligosaccharides (kestose, nystose and the like),
maltotetraol, maltotriol, malto-oligosaccharides (maltotriose,
maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the
like), starch, inulin, inulo-oligosaccharides, lactulose,
melibiose, raffinose, ribose, isomerized liquid sugars such as high
fructose corn syrups, coupling sugars, and soybean
oligosaccharides. Additionally, the carbohydrates as used herein
may be in either the D- or L-configuration.
[0138] The starting composition may be synthetic or purified
(partially or entirely), commercially available or prepared.
[0139] In one embodiment, the starting composition is glycerol.
[0140] In another embodiment, the starting composition is
glucose.
[0141] In still another embodiment, the starting composition is
sucrose.
[0142] In yet another embodiment, the starting composition is
starch.
[0143] In another embodiment, the starting composition is
maltodextrin.
[0144] In another embodiment, the starting composition is steviol
glycoside(s).
[0145] The organic compound(s) of starting composition serve as a
substrate(s) for the production of the target steviol glycoside(s),
as described herein.
[0146] Target Steviol Glycoside
[0147] The target steviol glycoside of the present method can be
any steviol glycoside that can be prepared by the process disclosed
herein. In one embodiment, the target steviol glycoside is selected
from the group consisting of steviolmonoside, steviolbioside,
rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside
G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A,
rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L,
rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside M2,
rebaudioside D, rebaudioside D2, rebaudioside N or rebaudioside 0,
or other glycoside of steviol.
[0148] In one embodiment, the target steviol glycoside is
stevioside. In another embodiment, the target steviol glycoside is
reb A. In still another embodiment, the target steviol glycoside is
reb E. In yet another embodiment, the target steviol glycoside is
reb D.
[0149] In yet another embodiment, the target steviol glycoside is
reb D2. In a further embodiment, the target steviol glycoside is
reb M. In a still further another embodiment, the target steviol
glycoside is reb M2.
[0150] The target steviol glycoside can be in any polymorphic or
amorphous form, including hydrates, solvates, anhydrous or
combinations thereof.
[0151] In one embodiment, the present invention is a biocatalytic
process for the production of reb D.
[0152] In yet another embodiment, the present invention is a
biocatalytic process for the production of reb D2.
[0153] In still another embodiment, the present invention is a
biocatalytic process for the production of reb M.
[0154] In a further embodiment, the present invention is a
biocatalytic process for the production of reb M2.
[0155] In one embodiment, the present invention is a biocatalytic
process for the production of reb I.
[0156] In yet another embodiment, the present invention is a
biocatalytic process for the production of reb E.
[0157] Optionally, the method of the present invention further
comprises separating the target steviol glycoside from the starting
composition. The target steviol glycoside can be separated by any
suitable method, such as, for example, crystallization, separation
by membranes, centrifugation, extraction, chromatographic
separation or a combination of such methods.
[0158] In particular embodiments, the process described herein
results in a highly purified target steviol glycoside composition.
The term "highly purified", as used herein, refers to a composition
having greater than about 80% by weight of the target steviol
glycoside on an anhydrous basis. In one embodiment, the highly
purified target steviol glycoside composition contains greater than
about 90% by weight of the target steviol glycoside on an anhydrous
basis, such as, for example, greater than about 91%, greater than
about 92%, greater than about 93%, greater than about 94%, greater
than about 95%, greater than about 96%, greater than about 97%,
greater than about 98% or greater than about 99% target steviol
glycoside content on a dry basis.
[0159] In one embodiment, when the target steviol glycoside is reb
M, the process described herein provides a composition having
greater than about 90% reb M content by weight on a dry basis. In
another particular embodiment, when the target steviol glycoside is
reb M, the process described herein provides a composition
comprising greater than about 95% reb M content by weight on a dry
basis.
[0160] In another embodiment, when the target steviol glycoside is
reb M2, the process described herein provides a composition having
greater than about 90% reb M2 content by weight on a dry basis. In
another particular embodiment, when the target steviol glycoside is
reb M2, the process described herein provides a composition
comprising greater than about 95% reb M2 content by weight on a dry
basis.
[0161] In yet another embodiment, when the target steviol glycoside
is reb D, the process described herein provides a composition
greater than about 90% reb D content by weight on a dry basis. In
another particular embodiment, when the target steviol glycoside is
reb D, the process described herein provides a composition
comprising greater than about 95% reb D content by weight on a dry
basis.
[0162] In still another embodiment, when the target steviol
glycoside is reb D2, the process described herein provides a
composition greater than about 90% reb D2 content by weight on a
dry basis. In another particular embodiment, when the target
steviol glycoside is reb D2, the process described herein provides
a composition comprising greater than about 95% reb D2 content by
weight on a dry basis.
[0163] In a further embodiment, when the target steviol glycoside
is reb A, the process described herein provides a composition
comprising greater than about 90% reb A content by weight on a dry
basis. In another particular embodiment, when the target steviol
glycoside is reb A, the process described herein provides a
composition comprising greater than about 95% reb A content by
weight on a dry basis.
[0164] In a still further embodiment, when the target steviol
glycoside is reb E, the process described herein provides a
composition comprising greater than about 90% reb E content by
weight on a dry basis. In another particular embodiment, when the
target steviol glycoside is reb E, the process described herein
provides a composition comprising greater than about 95% reb E
content by weight on a dry basis.
[0165] In one embodiment, when the target steviol glycoside is reb
I, the process described herein provides a composition comprising
greater than about 90% reb I content by weight on a dry basis. In
another particular embodiment, when the target steviol glycoside is
reb I, the process described herein provides a composition
comprising greater than about 95% reb I content by weight on a dry
basis.
[0166] In yet a further embodiment, when the target steviol
glycoside is stevioside, the process described herein provides a
composition comprising greater than about 90% stevioside content by
weight on a dry basis. In another particular embodiment, when the
target steviol glycoside is stevioside, the process described
herein provides a composition comprising greater than about 95%
stevioside content by weight on a dry basis.
[0167] Microorganism
[0168] In one embodiment of present invention, a microorganism is
contacted with the starting composition to produce a composition
comprising the target steviol glycoside. The microorganism can be
any microorganism possessing biocatalysts suitable for converting
the starting composition to the target steviol glycoside. These
biocatalysts are encoded within the microorganism's genome.
[0169] In one embodiment the microoganism may be, for example, E.
coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp.,
Yarrowia sp. etc.
[0170] The biocatalysts can be located on the surface and/or inside
the cell of the microorganism.
[0171] The biocatalysts can be separated from the microorganism and
used for conversion of starting composition to target steviol
glycoside(s). The separation can be achieved by any means known to
art, including but not limited to lysis of microbial cells,
centrifugation, filtration.
[0172] The biocatalysts can be excreted from the microorganism
(extracellular enzymes) and used for conversion of starting
composition to target steviol glycoside(s).
[0173] In one embodiment, the biocatalysts are steviol biosynthesis
enzymes and UDP-glycosyltransferases (UGTs), or their variants,
having greater than 75% amino-acid sequence identity.
[0174] The steviol biosynthesis can be any steviol biosynthesis
enzyme, or its variant, having greater than 75% amino-acid sequence
identity.
[0175] In one embodiment the steviol biosynthesis enzymes include
mevalonate (MVA) pathway enzymes, or their variants, having greater
than 75% amino-acid sequence identity.
[0176] In another embodiment the steviol biosynthesis enzymes
include non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway
(MEP/DOXP) enzymes, or their variants, having greater than 75%
amino-acid sequence identity.
[0177] In one embodiment, the steviol biosynthesis enzymes are
selected from the group including geranylgeranyl diphosphate
synthase, copalyl diphosphate synthase, kaurene synthase, kaurene
oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthetase,
deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose
5-phosphate reductoisomerase (DXR),
4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS),
4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK),
4-diphosphocytidyl-2-C-methyl-D-erythritol 2,4-cyclodiphosphate
synthase (MCS), l-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate
synthase (HDS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate
reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA
reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate
pyrophosphate decarboxylase, cytochrome P450 reductase, etc., or
their variants, having greater than 75% amino-acid sequence
identity.
[0178] The UDP-glucosyltransferase can be any
UDP-glucosyltransferase capable of adding at least one glucose unit
to the steviol and or steviol glycoside substrate to provide the
target steviol glycoside.
[0179] In one embodiment, the microorganism is free. In another
embodiment, the microorganism is immobilized. For example, the
microorganism may be immobilized to a solid support made from
inorganic or organic materials. Non-limiting examples of solid
supports suitable to immobilize the microorganism include
derivatized cellulose or glass, ceramics, metal oxides or
membranes. The microorganism may be immobilized to the solid
support, for example, by covalent attachment, adsorption,
cross-linking, entrapment or encapsulation.
[0180] In one embodiment the microorganism is in aqueous medium,
comprising water, and various components selected form group
including carbon sources, energy sources, nitrogen sources,
microelements, vitamins, nucleosides, nucleoside phosphates,
nucleoside diphosphates, nucleoside triphosphates, organic and
inorganic salts, organic and mineral acids, bases etc. Carbon
sources include glycerol, glucose, carbon dioxide, carbonates,
bicarbonates. Nitrogen sources can include nitrates, nitrites,
amino acids, peptides, peptones, or proteins.
[0181] In a particular embodiment, the medium comprises buffer.
Suitable buffers include, but are not limited to, PIPES buffer,
acetate buffer and phosphate buffer. In a particular embodiment,
the medium comprises phosphate buffer.
[0182] In one embodiment, the medium can also include an organic
solvent.
[0183] In one embodiment, the UDP-glucosyltransferase is any
UDP-glucosyltransferase capable of adding at least one glucose unit
to rubusoside, thereby producing stevioside. The
UDP-glucosyltransferase may be, for example, UGT91D2 or UGT91D2
variant, having greater than 75% amino-acid sequence identity with
UGT91D2.
[0184] In another embodiment, the UDP-glucosyltransferase is any
UDP-glucosyltransferase capable of adding at least one glucose unit
to rubusoside, thereby producing rebaudioside E. The
UDP-glucosyltransferase may be, for example, UGTSL2 or UGTSL2
variant, having greater than 75% amino-acid sequence identity with
UGTSL2.
[0185] In still another embodiment, the UDP-glucosyltransferase is
any UDP-glucosyltransferase capable of adding at least one glucose
unit to rebaudioside E, thereby producing rebaudioside D. The
UDP-glucosyltransferase may be, for example, UGT76G1 or UGT76G1
variant, having greater than 75% amino-acid sequence identity with
UGT76G1.
[0186] In yet embodiment, the UDP-glucosyltransferase is any
UDP-glucosyltransferase capable of adding at least one glucose unit
to stevioside, thereby producing rebaudioside A. The
UDP-glucosyltransferase may be, for example, UGT76G1 or UGT76G1
variant, having greater than 75% amino-acid sequence identity with
UGT76G1.
[0187] In a further embodiment, the UDP-glucosyltransferase is any
UDP-glucosyltransferase capable of adding at least one glucose unit
to rebaudioside A, thereby producing rebaudioside D and/or
rebaudioside D2 and/or rebaudioside M2. The UDP-glucosyltransferase
may be, for example, UGT91D2 or UGTSL2 or their variant, having
greater than 75% amino-acid sequence identity with UGT91D2 or
UGTSL2.
[0188] In yet another embodiment, the UDP-glucosyltransferase is
any UDP-glucosyltransferase capable of adding at least one glucose
unit to rebaudioside I to form rebaudioside M. In a particular
embodiment, the UDP-glucosyltransferase is UGTSL or UGTSL variant,
having greater than 75% amino-acid sequence identity with
UGTSL.
[0189] In yet another embodiment, the UDP-glucosyltransferase is
any UDP-glucosyltransferase capable of adding at least two glucose
units to rebaudioside E to form rebaudioside M. In a particular
embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT76G1
variant, having greater than 75% amino-acid sequence identity with
UGT76G1.
[0190] In another embodiment, the UDP-glucosyltransferase capable
of adding at least one glucose unit to produce target steviol
glycoside, has greater than 75% amino-acid sequence identity with
at least one enzyme selected from the following listing of GenInfo
identifier numbers, preferably from the group presented in Table 1,
and more preferably the group presented in Table 2.
TABLE-US-00002 397567 30680413 115480946 147798902 218193594
225443294 454245 32816174 116310259 147811764 218193942 225444853
1359905 32816178 116310985 147827151 219885307 225449296 1685003
34393978 116788066 147836230 222615927 225449700 1685005 37993665
116788606 147839909 222619587 225454338 2191136 37993671 116789315
147846163 222623142 225454340 2501497 37993675 119394507 147855977
222625633 225454342 2911049 39104603 119640480 148905778 222625635
225454473 4218003 41469414 122209731 148905999 222636620 225454475
4314356 41469452 125526997 148906835 222636621 225458362 13492674
42566366 125534279 148907340 222636628 225461551 13492676 42570280
125534461 148908935 222636629 225461556 15217773 42572855 125540090
148909182 224053242 225461558 15217796 44890129 125541516 148909920
224053386 225469538 15223396 46806235 125545408 148910082 224055535
225469540 15223589 50284482 125547340 148910154 224056138 226316457
15227766 51090402 125547520 148910612 224056160 226492603 15230017
51090594 125554547 148910769 224067918 226494221 15231757 52839682
125557592 156138791 224072747 226495389 15234056 56550539 125557593
156138797 224080189 226495945 15234195 62734263 125557608 156138799
224091845 226502400 15234196 62857204 125559566 156138803 224094703
226507980 15238503 62857206 125563266 165972256 224100653 226531147
15239523 62857210 125571055 168016721 224100657 226532094 15239525
62857212 125579728 171674071 224101569 238477377 15239543 75265643
125588307 171906258 224103105 240254512 15239937 75285934 125589492
183013901 224103633 242032615 15240305 75288884 125599469 183013903
224103637 242032621 15240534 77550661 125601477 186478321 224109218
242038423 15982889 77556148 126635837 187373030 224114583 242043290
18086351 82791223 126635845 187373042 224116284 242044836 18418378
83778990 126635847 190692175 224120552 242051252 18418380 89953335
126635863 194701936 224121288 242056217 18418382 110741436
126635867 195620060 224121296 242056219 19743740 110743955
126635883 209954691 224121300 242056663 19911201 115438196
126635887 209954719 224130358 242059339 20149064 115438785
133874210 209954725 224140703 242059341 20260654 115441237
133874212 209954733 224143404 242060922 21435782 115454819
145358033 210063105 224143406 242067411 21553613 115456047
147772508 210063107 224144306 242067413 21593514 115457492
147776893 212275846 224285244 242076258 22759895 115459312
147776894 216296854 225431707 242076396 23955910 115464719
147776895 217074506 225435532 242084750 26452040 115471069
147786916 218185693 225436321 242091005 28393204 115471071
147798900 218187075 225440041 242095206 30679796 115474009
147798901 218189427 225441116 242345159 242345161 297724601
326492035 356523945 357140904 359486938 255536859 297725463
326493430 356523957 357165849 359487055 255538228 297728331
326500410 356523959 357165852 359488135 255541676 297738632
326506816 356523961 357168415 359488708 255547075 297745347
326507826 356523963 357437837 359493630 255552620 297745348
326508394 356524387 357442755 359493632 255552622 297795735
326509445 356524403 357442757 359493634 255555343 297796253
326511261 356527181 357445729 359493636 255555361 297796257
326511866 356533209 357445731 359493815 255555363 297796261
326512412 356533852 357445733 359495856 255555365 297797587
326517673 356534718 357446799 359495858 255555369 297798502
326518800 356535480 357446805 359495869 255555373 297799226
326521124 356542996 357452779 359495871 255555377 297805988
326525567 356543136 357452781 359497638 255556812 297807499
326525957 356543932 357452783 359807261 255556818 297809125
326526607 356549841 357452787 374256637 255563008 297809127
326527141 356549843 357452789 377655465 255564074 297811403
326530093 356554358 357452791 378405177 255564531 297820040
326534036 356554360 357452797 378829085 255572878 297821483
326534312 356558606 357452799 387135070 255577901 297825217
332071132 356560333 357470367 387135072 255583249 297832276
339715876 356560599 357472193 387135078 255583253 297832280
342306012 356560749 357472195 387135092 255583255 297832518
342306016 356566018 357474295 387135094 255585664 297832520
343457675 356566169 357474493 387135098 255585666 297840825
343457677 356566173 357474497 387135100 255634688 297840827
350534960 356567761 357474499 387135134 255644801 297847402
356498085 356574704 357490035 387135136 255645821 297849372
356499771 356576401 357493567 387135174 255647456 300078590
356499777 356577660 357497139 387135176 255648275 300669727
356499779 357114993 357497581 387135184 260279126 302142947
356501328 357115447 357497671 387135186 260279128 302142948
356502523 357115451 357500579 387135188 261343326 302142950
356503180 357115453 357504663 387135190 283132367 302142951
356503184 357116080 357504691 387135192 283362112 302765302
356503295 357116928 357504699 387135194 289188052 302796334
356504436 357117461 357504707 387135282 295841350 302811470
356504523 357117463 357505859 387135284 296088529 302821107
356504765 357117829 357510851 387135294 296090415 302821679
356511113 357117839 357516975 387135298 296090524 319759260
356515120 357125059 359477003 387135300 296090526 319759266
356517088 357126015 359477998 387135302 297599503 320148814
356520732 357134488 359478043 387135304 297601531 326489963
356522586 357135657 359478286 387135312 297611791 326490273
356522588 357138503 359484299 387135314 297722841 326491131
356522590 357139683 359486936 387135316 387135318 449440433
460376293 460413408 462423864 475546199 387135320 449445896
460378310 460416351 470101924 475556485 387135322 449446454
460380744 462394387 470102280 475559699 387135324 449447657
460381726 462394433 470102858 475578293 387135326 449449002
460382093 462394557 470104211 475591753 387135328 449449004
460382095 462395646 470104264 475593742 388493506 449449006
460382754 462395678 470104266 475612072 388495496 449451379
460384935 462396388 470106317 475622476 388498446 449451589
460384937 462396389 470106357 475622507 388499220 449451591
460385076 462396419 470115448 475623787 388502176 449451593
460385872 462396542 470130404 482550481 388517521 449453712
460386018 462397507 470131550 482550499 388519407 449453714
460389217 462399998 470136482 482550740 388521413 449453716
460394872 462400798 470136484 482550999 388827901 449453732
460396139 462401217 470136488 482552352 388827903 449457075
460397862 462402118 470136492 482554970 388827907 449467555
460397864 462402237 470137933 482555336 388827909 449468742
460398541 462402284 470137937 482555478 388827913 449495638
460403139 462402416 470140422 482556454 393887637 449495736
460403141 462404228 470140426 482557289 393887646 449499880
460403143 462406358 470140908 482558462 393887649 449502786
460403145 462408262 470141232 482558508 393990627 449503471
460405998 462409325 470142008 482558547 397746860 449503473
460407578 462409359 470142010 482561055 397789318 449515857
460407590 462409777 470142012 482561555 413924864 449518643
460409128 462411467 470143607 482562795 414590349 449519559
460409134 462414311 470143939 482562850 414590661 449522783
460409136 462414416 470145404 482565074 414591157 449524530
460409459 462414476 473923244 482566269 414879558 449524591
460409461 462415526 474114354 482566296 414879559 449528823
460409463 462415603 474143634 482566307 414879560 449528825
460409465 462415731 474202268 482568689 414888074 449534021
460409467 462416307 474299266 482570049 431812559 460365546
460410124 462416920 474363119 482570572 449432064 460366882
460410126 462416922 474366157 482575121 449432066 460369823
460410128 462416923 474429346 449433069 460369829 460410130
462416924 475432777 449436944 460369831 460410132 462417401
475473002 449438665 460369833 460410134 462419769 475489790
449438667 460370755 460410213 462420317 475511330 449440431
460374714 460411200 462423366 475516200
TABLE-US-00003 TABLE 1 GI number Accession Origin 190692175
ACE87855.1 Stevia rebaudiana 41469452 AAS07253.1 Oryza sativa
62857204 BAD95881.1 Ipomoea nil 62857206 BAD95882.1 Ipomoea
purperea 56550539 BAD77944.1 Bellis perennis 115454819
NP_001051010.1 Oryza sativa Japonica Group 115459312 NP_001053256.1
Oryza sativa Japonica Group 115471069 NP_001059133.1 Oryza sativa
Japonica Group 115471071 NP_001059134.1 Oryza sativa Japonica Group
116310985 CAH67920.1 Oryza sativa Indica Group 116788066 ABK24743.1
Picea sitchensis 122209731 Q2V6J9.1 Fragaria .times. ananassa
125534461 EAY81009.1 Oryza sativa Indica Group 125559566 EAZ05102.1
Oryza sativa Indica Group 125588307 EAZ28971.1 Oryza sativa
Japonica Group 148907340 ABR16806.1 Picea sitchensis 148910082
ABR18123.1 Picea sitchensis 148910612 ABR18376.1 Picea sitchensis
15234195 NP_194486.1 Arabidopsis thaliana 15239523 NP_200210.1
Arabidopsis thaliana 15239937 NP_196793.1 Arabidopsis thaliana
1685005 AAB36653.1 Nicotiana tabacum 183013903 ACC38471.1 Medicago
truncatula 186478321 NP_172511.3 Arabidopsis thaliana 187373030
ACD03249.1 Avena strigosa 194701936 ACF85052.1 Zea mays 19743740
AAL92461.1 Solanum lycopersicum 212275846 NP_001131009.1 Zea mays
222619587 EEE55719.1 Oryza sativa Japonica Group 224055535
XP_002298527.1 Populus trichocarpa 224101569 XP_002334266.1 Populus
trichocarpa 224120552 XP_002318358.1 Populus trichocarpa 224121288
XP_002330790.1 Populus trichocarpa 225444853 XP_002281094 Vitis
vinifera 225454342 XP_002275850.1 Vitis vinifera 225454475
XP_002280923.1 Vitis vinifera 225461556 XP_002285222 Vitis vinifera
225469540 XP_002270294.1 Vitis vinifera 226495389 NP_001148083.1
Zea mays 226502400 NP_001147674.1 Zea mays 238477377 ACR43489.1
Triticum aestivum 240254512 NP_565540.4 Arabidopsis thaliana
2501497 Q43716.1 Petunia .times. hybrida 255555369 XP_002518721.1
Ricinus communis 26452040 BAC43110.1 Arabidopsis thaliana 296088529
CBI37520.3 Vitis vinifera 297611791 NP_001067852.2 Oryza sativa
Japonica Group 297795735 XP_002865752.1 Arabidopsis lyrata subsp.
lyrata 297798502 XP_002867135.1 Arabidopsis lyrata subsp. lyrata
297820040 XP_002877903.1 Arabidopsis lyrata subsp. lyrata 297832276
XP_002884020.1 Arabidopsis lyrata subsp. lyrata 302821107
XP_002992218.1 Selaginella moellendorffii 30680413 NP_179446.2
Arabidopsis thaliana 319759266 ADV71369.1 Pueraria montana var.
lobata 326507826 BAJ86656.1 Hordeum vulgare subsp. Vulgare
343457675 AEM37036.1 Brassica rapa subsp. oleifera 350534960
NP_001234680.1 Solanum lycopersicum 356501328 XP_003519477.1
Glycine max 356522586 XP_003529927.1 Glycine max 356535480
XP_003536273.1 Glycine max 357445733 XP_003593144.1 Medicago
truncatula 357452783 XP_003596668.1 Medicago truncatula 357474493
XP_003607531.1 Medicago truncatula 357500579 XP_003620578.1
Medicago truncatula 357504691 XP_003622634.1 Medicago truncatula
359477998 XP_003632051.1 Vitis vinifera 359487055 XP_002271587
Vitis vinifera 359495869 XP_003635104.1 Vitis vinifera 387135134
AFJ52948.1 Linum usitatissimum 387135176 AFJ52969.1 Linum
usitatissimum 387135192 AFJ52977.1 Linum usitatissimum 387135282
AFJ53022.1 Linum usitatissimum 387135302 AFJ53032.1 Linum
usitatissimum 387135312 AFJ53037.1 Linum usitatissimum 388519407
AFK47765.1 Medicago truncatula 393887646 AFN26668.1 Barbarea
vulgaris subsp. arcuata 414888074 DAA64088.1 Zea mays 42572855
NP_974524.1 Arabidopsis thaliana 449440433 XP_004137989.1 Cucumis
sativus 449446454 XP_004140986.1 Cucumis sativus 449449004
XP_004142255.1 Cucumis sativus 449451593 XP_004143546.1 Cucumis
sativus 449515857 XP_004164964.1 Cucumis sativus 460382095
XP_004236775.1 Solanum lycopersicum 460409128 XP_004249992.1
Solanum lycopersicum 460409461 XP_004250157.1 Solanum lycopersicum
460409465 XP_004250159.1 Solanum lycopersicum 462396388 EMJ02187.1
Prunus persica 462402118 EMJ07675.1 Prunus persica 462409359
EMJ14693.1 Prunus persica 462416923 EMJ21660.1 Prunus persica
46806235 BAD17459.1 Oryza sativa Japonica Group 470104266
XP_004288529.1 Fragaria vesca subsp. vesca 470142008 XP_004306714.1
Fragaria vesca subsp. vesca 475432777 EMT01232.1 Aegilops tauschii
51090402 BAD35324.1 Oryza sativa Japonica Group
TABLE-US-00004 TABLE 2 GI number Accession Origin 460409128
XP.004249992.1 Solanum lycopersicum 460386018 XP.004238697.1
Solanum lycopersicum 460409134 XP.004249995.1 Solanum lycopersicum
460410132 XP.004250485.1 Solanum lycopersicum 460410130
XP.004250484.1 Solanum lycopersicum 460410128 XP.004250483.1
Solanum lycopersicum 460378310 XP.004234916.1 Solanum lycopersicum
209954733 BAG80557.1 Lycium barbarum 209954725 BAG80553.1 Lycium
barbarum
[0191] In yet another embodiment, the UDP-glucosyltransferase is
any UDP-glucosyltransferase capable of adding at least one glucose
unit to rebaudioside D to form rebaudioside M and/or rebaudioside
M2. The UDP-glucosyltransferase may be, for example, UGT76G1 or
UGT76G1 variant, having greater than 75% amino-acid sequence
identity with UGT76G1.
[0192] Optionally, the method of the present invention further
comprises recycling UDP to provide UDP-glucose. In one embodiment,
the method comprises recycling UDP by providing a recycling
catalyst, i.e., a biocatalyst capable of UDP-glucose
overproduction, and a recycling substrate, such that the conversion
of the substrate steviol glycoside to the target steviol glycoside
is carried out using catalytic amounts of UDP-glucosyltransferase
and UDP-glucose (FIG. 3).
[0193] In one embodiment, the UDP-glucose recycling catalyst is
sucrose synthase.
[0194] In one embodiment, the recycling substrate is sucrose.
[0195] Optionally, the method of the present invention further
comprises hydrolysis of 1,6-.beta.-glucosidic linkages in reb D2
and/or reb M2. In one embodiment, the method comprises hydrolysis
of 1,6-.beta.-glucosidic linkages in reb D2 and/or reb M2 by
providing a .beta.-glucosidase.
[0196] In one embodiment .beta.-glucosidase is provided together
with UDP-recycling biocatalyst and UGTs to minimize the content of
reb D2 and/or reb M2 in final reaction mixture and maximize the
yield of reb M.
[0197] In a particular embodiment to minimize the content of reb D2
and/or reb M2 in final reaction mixture and maximize the yield of
reb M, .beta.-glucosidase is provided together with UDP-recycling
biocatalyst, UGT76G1 and UGTSL2, or their variants having greater
than 75% amino-acid sequence identity with UGT76G1 or UGTSL2.
[0198] The target steviol glycoside is optionally purified from the
resulting composition. Purification of the target steviol glycoside
from the reaction medium can be achieved by any suitable method to
provide a highly purified target steviol glycoside composition.
Suitable methods include crystallization, separation by membranes,
centrifugation, extraction (liquid or solid phase), chromatographic
separation, HPLC (preparative or analytical) or a combination of
such methods.
[0199] Compounds and Methods
[0200] The present invention also provides isolated and highly
purified reb D2. Reb D2 is an isomer of reb D and has the following
structure:
##STR00006##
13-[(2-O-.beta.-D-glucopyranosyl-3-O-.beta.-D-glucopyranosyl-.beta.-D-glu-
copyranosyl)oxy] ent-kaur-16-en-19-oic
acid-[(6-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)
ester]
[0201] In another embodiment, the present invention provides reb D2
having a purity greater than about 95% by weight on an anhydrous
basis, such as, for example, greater than about 96% by weight,
greater than about 97% by weight, greater than about 98% by weight
or greater than about 99% by weight.
[0202] In still another embodiment, the present invention provides
reb D2 having a purity greater than about 95% by weight in a
steviol glycoside mixture, such as, for example, greater than about
96% by weight, greater than about 97% by weight, greater than about
98% by weight or greater than about 99% by weight.
[0203] The present invention also provides compositions comprising
reb D2.
[0204] In one embodiment, the present invention provides a method
for preparing reb D2 comprising: [0205] a. contacting a starting
composition comprising reb A with an enzyme capable of transforming
reb A to reb D2, UDP-glucose, and optionally UDP-glucose recycling
enzymes, to produce a composition comprising reb D2; and [0206] b.
isolating the composition comprising reb D2.
[0207] In some embodiments, the enzyme capable of transforming reb
A to reb D2 is a UDP-glucosyltransferase, such as, for example,
UGT91D2, UGTSL, UGTSL_Sc, UGTSL2 (GI No. 460410132 version
XP_004250485.1), GI No. 460409128 (UGTSL) version XP_004249992.1,
GI No. 115454819 version NP_001051010.1, GI No. 187373030, version
ACD03249.1. GI No. 222619587 version EEE55719.1, GI No. 297795735
version XP_002865752.1 or EUGT11.
[0208] The enzyme capable of transforming reb A to reb D2 can be
immobilized or provided in the form of a recombinant
microorganism.
[0209] In one embodiment, the enzyme is immobilized. In another
embodiment, the enzyme is provided in the form of a recombinant
microorganism.
[0210] In one embodiment, the microorganism is free. In another
embodiment, the microorganism is immobilized. For example, the
microorganism may be immobilized to a solid support made from
inorganic or organic materials. Non-limiting examples of solid
supports suitable to immobilize the microorganism include
derivatized cellulose or glass, ceramics, metal oxides or
membranes. The microorganism may be immobilized to the solid
support, for example, by covalent attachment, adsorption,
cross-linking, entrapment or encapsulation.
[0211] Suitable microorganisms include, but are not limited to, E.
coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp.,
Yarrowia sp.
[0212] In one embodiment the microorganism is in an aqueous medium,
comprising water, and various components selected form group
including carbon sources, energy sources, nitrogen sources,
microelements, vitamins, nucleosides, nucleoside phosphates,
nucleoside diphosphates, nucleoside triphosphates, organic and
inorganic salts, organic and mineral acids, bases etc. Carbon
sources include glycerol, glucose, carbon dioxide, carbonates,
bicarbonates. Nitrogen sources can include nitrates, nitrites,
amino acids, peptides, peptones, or proteins.
[0213] In a particular embodiment, the medium comprises buffer.
Suitable buffers include, but are not limited to, PIPES buffer,
acetate buffer and phosphate buffer. In a particular embodiment,
the medium comprises phosphate buffer.
[0214] In one embodiment the medium can also include an organic
solvent.
[0215] In a particular embodiment, the enzyme is a
UDP-glucosyltransferase capable of transforming reb A to reb D2 and
is contained in E. coli.
[0216] In a more particular embodiment, the enzyme is selected from
UGT91D2, UGTSL, UGTSL_Sc, UGTSL2 (GI No. 460410132 version
XP_004250485.1), GI No. 460409128 (UGTSL) verison XP_004249992.1,
GI No. 115454819 version NP_001051010.1, GI No. 187373030, version
ACD03249.1. GI No. 222619587 version EEE55719.1, GI No. 297795735
version XP_002865752.1 or EUGT11 and is contained in E. coli.
[0217] In a still more particular embodiment, the enzyme is UGTSL2
and is contained in E. coli.
[0218] Isolation of reb D2 from the reaction medium can be achieved
by any suitable method to provide a composition comprising reb D2.
Suitable methods include, but are not limited to, lysis,
crystallization, separation by membranes, centrifugation,
extraction (liquid or solid phase), chromatographic separation,
HPLC (preparative or analytical) or a combination of such methods.
In a particular embodiment, isolation can be achieved by lysis and
centrifugation.
[0219] In some embodiments, isolation may result in a reb D2 purity
less than about 95% by weight on an anhydrous basis, and the
composition may contain, e.g., steviol glycosides and/or residual
reaction products. The composition comprising reb D2 can be further
purified to provide highly purified reb D2, i.e. reb D2 having a
purity greater than about 95% by weight on an anhydrous basis. In
some embodiments, the compositions comprising reb D2 can be further
purified to provide reb D2 having a purity greater than about 96%,
greater than about 97%, greater than about 98% or greater than
about 99% by weight on an anhydrous basis.
[0220] Purification can be affected by any means known to one of
skill in the art including, but not limited to, crystallization,
separation by membranes, centrifugation, extraction (liquid or
solid phase), chromatographic separation, HPLC (preparative or
analytical) or a combination of such methods. In a particular
embodiment, HPLC is used to purify reb D2. In a more particular
embodiment, semi-preparative HPLC is used to purify reb D2.
[0221] For example, a two-step semi-preparative HPLC purification
can be used. The first step utilizes a C18 column with a mobile
phase containing A (25% MeCN in water) and B (30% MeCN in water)
with the following gradient:
TABLE-US-00005 Time (min) % A % B 0.0-5.0 100 0 20 20 80 25 20 80
30 100 0
[0222] The secondary step utilizes the same column and conditions,
but with only an isocratic mobile phase: 20% MeCN in water.
[0223] Those of skill in the art will recognize that the particular
column, mobile phases, injection volumes and other HPLC parameters
can vary.
[0224] In one embodiment, the present invention provides isolated
and highly purified reb M2. Reb M2 is an isomer of reb M and has
the following structure:
##STR00007##
13-[(2-O-.beta.-D-glucopyranosyl-3-O-.beta.-D-glucopyranosyl-.beta.-D-glu-
copyranosyl)oxy] ent-kaur-16-en-19-oic
acid-[(2-O-.beta.-D-glucopyranosyl-6-O-.beta.-D-glucopyranosyl-.beta.-D-g-
lucopyranosyl) ester]
[0225] In another embodiment, the present invention provides reb M2
having a purity greater than about 95% by weight on an anhydrous
basis, such as, for example, greater than about 96% by weight,
greater than about 97% by weight, greater than about 98% by weight
or greater than about 99% by weight.
[0226] In still another embodiment, the present invention provides
reb M2 having a purity greater than about 95% by weight in a
steviol glycoside mixture, such as, for example, greater than about
96% by weight, greater than about 97% by weight, greater than about
98% by weight or greater than about 99% by weight.
[0227] In yet another embodiment, the present invention provides
reb M2 having a purity greater than about 95% by weight in a stevia
extract, such as, for example, greater than about 96% by weight,
greater than about 97% by weight, greater than about 98% by weight
or greater than about 99% by weight.
[0228] The present invention also provides compositions comprising
reb M2.
[0229] It has been found that reb M2 is produced during
biotransformation of reb A to reb D. As noted above,
biotransformation of reb A to reb D also produces reb D2.
Accordingly, in one embodiment, the present invention provides a
method for preparing reb M2 comprising: [0230] a. contacting a
starting composition comprising reb A and/or reb D2 with an enzyme
capable of transforming reb A and/or reb D2 to reb M2, UDP-glucose,
and optionally UDP-glucose recycling enzymes to produce a
composition comprising reb M2; and [0231] b. isolating a
composition comprising reb M2.
[0232] Not wishing to be bound by theory, it is currently believed
that the pathway begins with transformation of reb A to reb D2,
followed by transformation of reb D2 to reb M2. Accordingly, In one
embodiment, the present invention provides a method for preparing
reb M2 comprising: [0233] a. contacting a starting composition
comprising reb D2 with an enzyme capable of transforming reb D2 to
reb M2, UDP-glucose, and optionally UDP-glucose recycling enzymes
to produce a composition comprising reb M2; and [0234] b. isolating
a composition comprising reb M2.
[0235] In yet another embodiment, a method for preparing reb M2
comprises: [0236] a. contacting a starting composition comprising
reb A with an enzyme capable of transforming reb A to reb D2,
UDP-glucose, and optionally UDP-glucose recycling enzymes to
produce a composition comprising reb D2; [0237] b. optionally,
isolating a composition comprising reb D2; [0238] c. contacting the
composition comprising reb D2 with an enzyme capable of
transforming reb D2 to reb M2, UDP-glucose, and optionally
UDP-glucose recycling enzymes to produce a composition comprising
reb M2; and [0239] d. isolating a composition comprising reb
M2.
[0240] The enzyme can be a UDP-glucosyltransferase, such as, for
example, UGT91D2, UGTSL, UGTSL_Sc, UGTSL2 (GI No. 460410132 version
XP_004250485.1), GI No. 460409128 (UGTSL) version XP_004249992.1,
GI No. 115454819 version NP_001051010.1, GI No. 187373030, version
ACD03249.1. GI No. 222619587 version EEE55719.1, GI No. 297795735
version XP_002865752.1 or EUGT11.
[0241] The enzyme can be immobilized or in a recombinant
microorganism.
[0242] In one embodiment, the enzyme is immobilized. In another
embodiment, the enzyme is in a recombinant microorganism.
[0243] In one embodiment, the microorganism is free. In another
embodiment, the microorganism is immobilized. For example, the
microorganism may be immobilized to a solid support made from
inorganic or organic materials. Non-limiting examples of solid
supports suitable to immobilize the microorganism include
derivatized cellulose or glass, ceramics, metal oxides or
membranes. The microorganism may be immobilized to the solid
support, for example, by covalent attachment, adsorption,
cross-linking, entrapment or encapsulation.
[0244] Suitable microorganisms include, but are not limited to, E.
coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp.,
Yarrowia sp.
[0245] In one embodiment the microorganism is in aqueous medium,
comprising water, and various components selected form group
including carbon sources, energy sources, nitrogen sources,
microelements, vitamins, nucleosides, nucleoside phosphates,
nucleoside diphosphates, nucleoside triphosphates, organic and
inorganic salts, organic and mineral acids, bases etc. Carbon
sources include glycerol, glucose, carbon dioxide, carbonates,
bicarbonates. Nitrogen sources can include nitrates, nitrites,
amino acids, peptides, peptones, or proteins.
[0246] In a particular embodiment, the medium comprises buffer.
Suitable buffers include, but are not limited to, PIPES buffer,
acetate buffer and phosphate buffer. In a particular embodiment,
the medium comprises phosphate buffer.
[0247] In one embodiment the medium can also include an organic
solvent.
[0248] In a particular embodiment, the enzyme is a
UDP-glucosyltransferase capable of transforming reb A and/or reb D2
to reb M2 and is contained in E. coli.
[0249] In a more particular embodiment, the enzyme is selected from
UGT91D2, UGTSL, UGTSL_Sc, UGTSL2 (GI No. 460410132 version
XP_004250485.1), GI No. 460409128 (UGTSL) verison XP_004249992.1,
GI No. 115454819 version NP 001051010.1, GI No. 187373030, version
ACD03249.1. GI No. 222619587 version EEE55719.1, GI No. 297795735
version XP_002865752.1 or EUGT11 and is contained in E. coli.
[0250] In a still more particular embodiment, the enzyme is UGTSL2
and is contained in E. coli.
[0251] Isolation of reb M2 from the reaction medium can be achieved
by any suitable method to provide a composition comprising reb M2.
Suitable methods include, but are not limited to, lysis,
crystallization, separation by membranes, centrifugation,
extraction (liquid or solid phase), chromatographic separation,
HPLC (preparative or analytical) or a combination of such methods.
In a particular embodiment, isolation can be achieved by lysis and
centrifugation.
[0252] In some embodiments, isolation may result in a reb M2 purity
less than about 95% by weight on an anhydrous basis, and the
composition may contain, e.g., steviol glycosides and/or residual
reaction products.
[0253] The composition comprising reb M2 can be further purified to
provide highly purified reb M2, i.e. reb M2 having a purity greater
than about 95% by weight on an anhydrous basis. In some
embodiments, the compositions comprising reb M2 can be further
purified to provide reb M2 having a purity greater than about 96%,
greater than about 97%, greater than about 98% or greater than
about 99% by weight on an anhydrous basis.
[0254] Purification can be affected by any means known to one of
skill in the art including, but not limited to, crystallization,
separation by membranes, centrifugation, extraction (liquid or
solid phase), chromatographic separation, HPLC (preparative or
analytical) or a combination of such methods. In a particular
embodiment, HPLC is used to purify reb M2. In a more particular
embodiment, semi-preparative HPLC is used to purify reb M2.
[0255] For example, a two-step semi-preparative HPLC purification
can be used. The first step utilizes a C18 column with a mobile
phase containing A (25% MeCN in water) and B (30% MeCN in water)
with the following gradient:
TABLE-US-00006 Time (min) % A % B 0.0-5.0 100 0 20 20 80 25 20 80
30 100 0
[0256] The secondary step utilizes the same column and conditions,
but with only an isocratic mobile phase: 20% MeCN in water.
[0257] Those of skill in the art will recognize that the particular
column, mobile phases, injection volumes and other HPLC parameters
can vary.
[0258] Purified steviol glycosides, prepared in accordance with the
present invention, may be used in a variety of consumable products
including, but not limited to, foods, beverages, pharmaceutical
compositions, tobacco products, nutraceutical compositions, oral
hygiene compositions, and cosmetic compositions.
[0259] The high purity reb M obtained in this invention, having a
molecular weight of 1291.29, a molecular formula of
C.sub.56H.sub.90O.sub.33, CAS registry number 1220616-44-3, and the
structure presented in FIG. 1, is in the form of a white and
odorless powder. The compound is about 200 times sweeter than sugar
when compared to a 10% sucrose solution. The infrared absorption
spectrum is shown in FIG. 4.
[0260] Other properties of the pure reb M compound include a
melting point of 249-250.degree. C., and a specific rotation of
[.alpha.].sub.D.sup.25-19.0.degree. in 50% ethanol (C=1.0). The
solubility of reb Min water is around 0.3%, and increases with an
increase in temperature.
[0261] Reb M is soluble in diluted solutions of methanol, ethanol,
n-propanol, and isopropanol. However, it is insoluble in acetone,
benzene, chloroform, and ether.
[0262] Reb M obtained in accordance with the present invention is
heat and pH-stable.
[0263] Highly purified target glycoside(s) particularly, reb D, reb
D2, reb M and/or reb M2 obtained according to this invention can be
used "as-is" or in combination with other sweeteners, flavors and
food ingredients.
[0264] Non-limiting examples of flavors include lime, lemon,
orange, fruit, banana, grape, pear, pineapple, mango, bitter
almond, cola, cinnamon, sugar, cotton candy and vanilla
flavors.
[0265] Non-limiting examples of other food ingredients include
flavors, acidulants, organic and amino acids, coloring agents,
bulking agents, modified starches, gums, texturizers,
preservatives, antioxidants, emulsifiers, stabilizers, thickeners
and gelling agents.
[0266] Highly purified target glycoside(s) particularly, reb D, reb
D2, reb M and/or reb M2 obtained according to this invention can be
prepared in various polymorphic forms, including but not limited to
hydrates, solvates, anhydrous, amorphous forms and/or mixtures
thereof.
[0267] Highly purified target steviol glycoside(s), particularly,
reb D, reb D2, reb M and/or reb M2 obtained according to this
invention may be incorporated as a high intensity natural sweetener
in foodstuffs, beverages, pharmaceutical compositions, cosmetics,
chewing gums, table top products, cereals, dairy products,
toothpastes and other oral cavity compositions, etc.
[0268] Highly purified target steviol glycoside(s), particularly,
reb D, reb D2, reb M and/or reb M2 as a sweetening compound may be
employed as the sole sweetener, or it may be used together with
other naturally occurring high intensity sweeteners such as
stevioside, reb A, reb B, reb C, reb D, reb E, reb F,
steviolbioside, dulcoside A, rubusoside, mogrosides, brazzein,
neohesperidin dihydrochalcone, glycyrrhizic acid and its salts,
thaumatin, perillartine, pernandulcin, mukuroziosides, baiyunoside,
phlomisoside-I, dimethyl-hexahydrofluorene-dicarboxylic acid,
abrusosides, periandrin, carnosiflosides, cyclocarioside,
pterocaryosides, polypodoside A, brazilin, hernandulcin,
phillodulcin, glycyphyl lin, phlorizin, trilobatin, di hydroflavon
ol, dihydroquercetin-3-acetate, neoastilibin, trans-cinnamaldehyde,
monatin and its salts, selligueain A, hematoxylin, monellin,
osladin, pterocaryoside A, pterocaryoside B, mabinlin, pentadin,
miraculin, curculin, neoculin, chlorogenic acid, cynarin, Luo Han
Guo sweetener, mogroside V, siamenoside and others.
[0269] In a particular embodiment, reb D2 and/or reb M2 can be used
together in a sweetener composition comprising a compound selected
from the group consisting of reb A, reb B, reb D, NSF-02, Mogroside
V, erythritol and combinations thereof.
[0270] Highly purified target steviol glycoside(s), particularly,
reb D, reb D2, reb M and/or reb M2 may also be used in combination
with synthetic high intensity sweeteners such as sucralose,
potassium acesulfame, aspartame, alitame, saccharin, neohesperidin
dihydrochalcone, cyclamate, neotame, dulcin, suosan advantame,
salts thereof, and the like.
[0271] Moreover, highly purified target steviol glycoside(s),
particularly, reb D, reb D2, reb M and/or reb M2 can be used in
combination with natural sweetener suppressors such as gymnemic
acid, hodulcin, ziziphin, lactisole, and others. Reb D, reb D2, reb
M and/or reb M2 may also be combined with various umami taste
enhancers. Reb D, reb D2, reb M and/or reb M2 can be mixed with
umami tasting and sweet amino acids such as glutamate, aspartic
acid, glycine, alanine, threonine, proline, serine, glutamate,
lysine and tryptophan.
[0272] Highly purified target steviol glycoside(s), particularly,
reb D, reb D2, reb M can be used in combination with one or more
additive selected from the group consisting of carbohydrates,
polyols, amino acids and their corresponding salts, poly-amino
acids and their corresponding salts, sugar acids and their
corresponding salts, nucleotides, organic acids, inorganic acids,
organic salts including organic acid salts and organic base salts,
inorganic salts, bitter compounds, flavorants and flavoring
ingredients, astringent compounds, proteins or protein
hydrolysates, surfactants, emulsifiers, flavonoids, alcohols,
polymers and combinations thereof.
[0273] Highly purified target steviol glycoside(s), particularly,
reb D, reb D2, reb M and/or reb M2 may be combined with polyols or
sugar alcohols. The term "polyol" refers to a molecule that
contains more than one hydroxyl group. A polyol may be a diol,
triol, or a tetraol which contain 2, 3, and 4 hydroxyl groups,
respectively. A polyol also may contain more than four hydroxyl
groups, such as a pentaol, hexaol, heptaol, or the like, which
contain 5, 6, or 7 hydroxyl groups, respectively. Additionally, a
polyol also may be a sugar alcohol, polyhydric alcohol, or
polyalcohol which is a reduced form of carbohydrate, wherein the
carbonyl group (aldehyde or ketone, reducing sugar) has been
reduced to a primary or secondary hydroxyl group. Examples of
polyols include, but are not limited to, erythritol, maltitol,
mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene
glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose,
reduced isomalto-oligosaccharides, reduced xylo-oligosaccharides,
reduced gentio-oligosaccharides, reduced maltose syrup, reduced
glucose syrup, hydrogenated starch hydrolyzates, polyglycitols and
sugar alcohols or any other carbohydrates capable of being reduced
which do not adversely affect the taste of the sweetener
composition.
[0274] Highly purified target steviol glycoside(s), particularly,
reb D, reb D2, reb M and/or reb M2 may be combined with reduced
calorie sweeteners such as D-tagatose, L-sugars, L-sorbose,
L-arabinose, and others.
[0275] Highly purified target steviol glycoside(s), particularly,
reb D, reb D2, reb M and/or reb M2 may also be combined with
various carbohydrates. The term "carbohydrate" generally refers to
aldehyde or ketone compounds substituted with multiple hydroxyl
groups, of the general formula (CH.sub.2O).sub.n, wherein n is
3-30, as well as their oligomers and polymers. The carbohydrates of
the present invention can, in addition, be substituted or
deoxygenated at one or more positions. Carbohydrates, as used
herein, encompass unmodified carbohydrates, carbohydrate
derivatives, substituted carbohydrates, and modified carbohydrates.
As used herein, the phrases "carbohydrate derivatives",
"substituted carbohydrate", and "modified carbohydrates" are
synonymous. Modified carbohydrate means any carbohydrate wherein at
least one atom has been added, removed, or substituted, or
combinations thereof. Thus, carbohydrate derivatives or substituted
carbohydrates include substituted and unsubstituted
monosaccharides, disaccharides, oligosaccharides, and
polysaccharides. The carbohydrate derivatives or substituted
carbohydrates optionally can be deoxygenated at any corresponding
C-position, and/or substituted with one or more moieties such as
hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino,
amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino,
alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl,
sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido,
phosphonyl, phosphinyl, phosphoryl, phosphino, thioester,
thioether, oximino, hydrazino, carbamyl, phospho, phosphonato, or
any other viable functional group provided the carbohydrate
derivative or substituted carbohydrate functions to improve the
sweet taste of the sweetener composition.
[0276] Examples of carbohydrates which may be used in accordance
with this invention include, but are not limited to, Psicose,
turanose, allose, tagatose, trehalose, galactose, rhamnose, various
cyclodextrins, cyclic oligosaccharides, various types of
maltodextrins, dextran, sucrose, glucose, ribulose, fructose,
threose, arabinose, xylose, lyxose, allose, altrose, mannose,
idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose,
isomaltulose, erythrose, deoxyribose, gulose, idose, talose,
erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin,
glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid,
glucono-lactone, abequose, galactosamine, beet oligosaccharides,
isomalto-oligosaccharides (isomaltose, isomaltotriose, panose and
the like), xylo-oligosaccharides (xylotriose, xylobiose and the
like), xylo-terminated oligosaccharides, gentio-oligosaccharides
(gentiobiose, gentiotriose, gentiotetraose and the like), sorbose,
nigero-oligosaccharides, palatinose oligosaccharides,
fructooligosaccharides (kestose, nystose and the like),
maltotetraol, maltotriol, malto-oligosaccharides (maltotriose,
maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the
like), starch, inulin, inulo-oligosaccharides, lactulose,
melibiose, raffinose, ribose, isomerized liquid sugars such as high
fructose corn syrups, coupling sugars, and soybean
oligosaccharides. Additionally, the carbohydrates as used herein
may be in either the D- or L-configuration.
[0277] Highly purified target steviol glycoside(s), particularly,
reb D, reb D2, reb M and/or reb M2 obtained according to this
invention can be used in combination with various physiologically
active substances or functional ingredients. Functional ingredients
generally are classified into categories such as carotenoids,
dietary fiber, fatty acids, saponins, antioxidants, nutraceuticals,
flavonoids, isothiocyanates, phenols, plant sterols and stanols
(phytosterols and phytostanols); polyols; prebiotics, probiotics;
phytoestrogens; soy protein; sulfides/thiols; amino acids;
proteins; vitamins; and minerals. Functional ingredients also may
be classified based on their health benefits, such as
cardiovascular, cholesterol-reducing, and anti-inflammatory.
Exemplary functional ingredients are provided in WO2013/096420, the
contents of which is hereby incorporated by reference.
[0278] Highly purified target steviol glycoside(s), particularly,
reb D, reb D2, reb M and/or reb M2 obtained according to this
invention may be applied as a high intensity sweetener to produce
zero calorie, reduced calorie or diabetic beverages and food
products with improved taste characteristics. It may also be used
in drinks, foodstuffs, pharmaceuticals, and other products in which
sugar cannot be used. In addition, highly purified target steviol
glycoside(s), particularly, reb D, reb D2, reb M and/or reb M2 can
be used as a sweetener not only for drinks, foodstuffs, and other
products dedicated for human consumption, but also in animal feed
and fodder with improved characteristics.
[0279] Examples of consumable products in which highly purified
target steviol glycoside(s), particularly, reb D, reb D2, reb M
and/or reb M2 may be used as a sweetening compound include, but are
not limited to, alcoholic beverages such as vodka, wine, beer,
liquor, and sake, etc.; natural juices; refreshing drinks;
carbonated soft drinks; diet drinks; zero calorie drinks; reduced
calorie drinks and foods; yogurt drinks; instant juices; instant
coffee; powdered types of instant beverages; canned products;
syrups; fermented soybean paste; soy sauce; vinegar; dressings;
mayonnaise; ketchups; curry; soup; instant bouillon; powdered soy
sauce; powdered vinegar; types of biscuits; rice biscuit; crackers;
bread; chocolates; caramel; candy; chewing gum; jelly; pudding;
preserved fruits and vegetables; fresh cream; jam; marmalade;
flower paste; powdered milk; ice cream; sorbet; vegetables and
fruits packed in bottles; canned and boiled beans; meat and foods
boiled in sweetened sauce; agricultural vegetable food products;
seafood; ham; sausage; fish ham; fish sausage; fish paste; deep
fried fish products; dried seafood products; frozen food products;
preserved seaweed; preserved meat; tobacco; medicinal products; and
many others. In principle it can have unlimited applications.
[0280] During the manufacturing of products such as foodstuffs,
drinks, pharmaceuticals, cosmetics, table top products, and chewing
gum, the conventional methods such as mixing, kneading,
dissolution, pickling, permeation, percolation, sprinkling,
atomizing, infusing and other methods may be used.
[0281] Moreover, the highly purified target steviol glycoside(s),
particularly, reb D, reb D2, reb M and/or reb M2 obtained in this
invention may be used in dry or liquid forms. In one embodiment, a
tabletop sweetener comprising reb D2 is provided. In another
embodiment, a tabletop sweetener comprising reb M2 is provided.
[0282] The highly purified target steviol glycoside can be added
before or after heat treatment of food products. The amount of the
highly purified target steviol glycoside(s), particularly, reb D,
reb D2, reb M and/or reb M2 depends on the purpose of usage. As
discussed above, it can be added alone or in combination with other
compounds.
[0283] The present invention is also directed to sweetness
enhancement in beverages using reb D2. The present invention is
also directed to sweetness enhancement in beverages containing reb
M2. Accordingly, the present invention provides a beverage
comprising a sweetener and reb D2 and/or reb M2 as a sweetness
enhancer, wherein reb D2 and/or reb M2 is present in a
concentration at or below their respective sweetness recognition
thresholds.
[0284] As used herein, the term "sweetness enhancer" refers to a
compound capable of enhancing or intensifying the perception of
sweet taste in a composition, such as a beverage. The term
"sweetness enhancer" is synonymous with the terms "sweet taste
potentiator," "sweetness potentiator," "sweetness amplifier," and
"sweetness intensifier."
[0285] The term "sweetness recognition threshold concentration," as
generally used herein, is the lowest known concentration of a sweet
compound that is perceivable by the human sense of taste, typically
around 1.0% sucrose equivalence (1.0% SE). Generally, the sweetness
enhancers may enhance or potentiate the sweet taste of sweeteners
without providing any noticeable sweet taste by themselves when
present at or below the sweetness recognition threshold
concentration of a given sweetness enhancer; however, the sweetness
enhancers may themselves provide sweet taste at concentrations
above their sweetness recognition threshold concentration. The
sweetness recognition threshold concentration is specific for a
particular enhancer and can vary based on the beverage matrix. The
sweetness recognition threshold concentration can be easily
determined by taste testing increasing concentrations of a given
enhancer until greater than 1.0% sucrose equivalence in a given
beverage matrix is detected. The concentration that provides about
1.0% sucrose equivalence is considered the sweetness recognition
threshold.
[0286] In some embodiments, sweetener is present in the beverage in
an amount from about 0.5% to about 12% by weight, such as, for
example, about 1.0% by weight, about 1.5% by weight, about 2.0% by
weight, about 2.5% by weight, about 3.0% by weight, about 3.5% by
weight, about 4.0% by weight, about 4.5% by weight, about 5.0% by
weight, about 5.5% by weight, about 6.0% by weight, about 6.5% by
weight, about 7.0% by weight, about 7.5% by weight, about 8.0% by
weight, about 8.5% by weight, about 9.0% by weight, about 9.5% by
weight, about 10.0% by weight, about 10.5% by weight, about 11.0%
by weight, about 11.5% by weight or about 12.0% by weight.
[0287] In a particular embodiment, the sweetener is present in the
beverage in an amount from about 0.5% of about 10%, such as for
example, from about 2% to about 8%, from about 3% to about 7% or
from about 4% to about 6% by weight. In a particular embodiment,
the sweetener is present in the beverage in an amount from about
0.5% to about 8% by weight. In another particular embodiment, the
sweetener is present in the beverage in an amount from about 2% to
about 8% by weight.
[0288] In one embodiment, the sweetener is a traditional caloric
sweetener. Suitable sweeteners include, but are not limited to,
sucrose, fructose, glucose, high fructose corn syrup and high
fructose starch syrup.
[0289] In another embodiment, the sweetener is erythritol.
[0290] In still another embodiment, the sweetener is a rare sugar.
Suitable rare sugars include, but are not limited to, D-allose,
D-psicose, L-ribose, D-tagatose, L-glucose, L-fucose, L-arbinose,
D-turanose, D-leucrose and combinations thereof.
[0291] It is contemplated that a sweetener can be used alone, or in
combination with other sweeteners.
[0292] In one embodiment, the rare sugar is D-allose. In a more
particular embodiment, D-allose is present in the beverage in an
amount of about 0.5% to about 10% by weight, such as, for example,
from about 2% to about 8%.
[0293] In another embodiment, the rare sugar is D-psicose. In a
more particular embodiment, D-psicose is present in the beverage in
an amount of about 0.5% to about 10% by weight, such as, for
example, from about 2% to about 8%.
[0294] In still another embodiment, the rare sugar is D-ribose. In
a more particular embodiment, D-ribose is present in the beverage
in an amount of about 0.5% to about 10% by weight, such as, for
example, from about 2% to about 8%.
[0295] In yet another embodiment, the rare sugar is D-tagatose. In
a more particular embodiment, D-tagatose is present in the beverage
in an amount of about 0.5% to about 10% by weight, such as, for
example, from about 2% to about 8%.
[0296] In a further embodiment, the rare sugar is L-glucose. In a
more particular embodiment, L-glucose is present in the beverage in
an amount of about 0.5% to about 10% by weight, such as, for
example, from about 2% to about 8%.
[0297] In one embodiment, the rare sugar is L-fucose. In a more
particular embodiment, L-fucose is present in the beverage in an
amount of about 0.5% to about 10% by weight, such as, for example,
from about 2% to about 8%.
[0298] In another embodiment, the rare sugar is L-arabinose. In a
more particular embodiment, L-arabinose is present in the beverage
in an amount of about 0.5% to about 10% by weight, such as, for
example, from about 2% to about 8%.
[0299] In yet another embodiment, the rare sugar is D-turanose. In
a more particular embodiment, D-turanose is present in the beverage
in an amount of about 0.5% to about 10% by weight, such as, for
example, from about 2% to about 8%.
[0300] In yet another embodiment, the rare sugar is D-leucrose. In
a more particular embodiment, D-leucrose is present in the beverage
in an amount of about 0.5% to about 10% by weight, such as, for
example, from about 2% to about 8%.
[0301] The addition of the sweetness enhancer at a concentration at
or below its sweetness recognition threshold increases the detected
sucrose equivalence of the beverage comprising the sweetener and
the sweetness enhancer compared to a corresponding beverage in the
absence of the sweetness enhancer. Moreover, sweetness can be
increased by an amount more than the detectable sweetness of a
solution containing the same concentration of the at least one
sweetness enhancer in the absence of any sweetener.
[0302] Accordingly, the present invention also provides a method
for enhancing the sweetness of a beverage comprising a sweetener
comprising providing a beverage comprising a sweetener and adding a
sweetness enhancer selected from reb D2, reb M2 or a combination
thereof, wherein reb D2 and reb M2 are present in a concentration
at or below their sweetness recognition thresholds.
[0303] Addition of reb D2 and/or reb M2 in a concentration at or
below the sweetness recognition threshold to a beverage containing
a sweetener may increase the detected sucrose equivalence from
about 1.0% to about 5.0%, such as, for example, about 1.0%, about
1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%,
about 4.5% or about 5.0%.
[0304] The following examples illustrate preferred embodiments of
the invention for the preparation of highly purified target steviol
glycoside(s), particularly, reb D, reb D2, reb M and/or reb M2. It
will be understood that the invention is not limited to the
materials, proportions, conditions and procedures set forth in the
examples, which are only illustrative.
Example 1
[0305] In-vivo production of UGT76G1
[0306] NcoI and NdeI restriction sides were added to the original
nucleic sequence as described in Genbank accession no. AAR06912.1.
After codon optimization the following nucleic sequence was
obtained:
TABLE-US-00007 CCATGGCCCATATGGAAAACAAAACCGAAACCACCGTTCGTCGTCGTCGC
CGTATTATTCTGTTTCCGGTTCCGTTTCAGGGTCATATTAATCCGATTCT
GCAGCTGGCAAATGTGCTGTATAGCAAAGGTTTTAGCATTACCATTTTTC
ATACCAATTTTAACAAACCGAAAACCAGCAATTATCCGCATTTTACCTTT
CGCTTTATTCTGGATAATGATCCGCAGGATGAACGCATTAGCAATCTGCC
GACACATGGTCCGCTGGCAGGTATGCGTATTCCGATTATTAACGAACATG
GTGCAGATGAACTGCGTCGTGAACTGGAACTGCTGATGCTGGCAAGCGAA
GAAGATGAAGAAGTTAGCTGTCTGATTACCGATGCACTGTGGTATTTTGC
ACAGAGCGTTGCAGATAGCCTGAATCTGCGTCGTCTGGTTCTGATGACCA
GCAGCCTGTTTAACTTTCATGCACATGTTAGCCTGCCGCAGTTTGATGAA
CTGGGTTATCTGGATCCGGATGATAAAACCCGTCTGGAAGAACAGGCAAG
CGGTTTTCCGATGCTGAAAGTGAAAGATATCAAAAGCGCCTATAGCAATT
GGCAGATTCTGAAAGAAATTCTGGGCAAAATGATTAAACAGACCAAAGCA
AGCAGCGGTGTTATTTGGAATAGCTTTAAAGAACTGGAAGAAAGCGAACT
GGAAACCGTGATTCGTGAAATTCCGGCACCGAGCTTTCTGATTCCGCTGC
CGAAACATCTGACCGCAAGCAGCAGCAGCCTGCTGGATCATGATCGTACC
GTTTTTCAGTGGCTGGATCAGCAGCCTCCGAGCAGCGTTCTGTATGTTAG
CTTTGGTAGCACCAGCGAAGTTGATGAAAAAGATTTTCTGGAAATTGCCC
GTGGTCTGGTTGATAGCAAACAGAGCTITCTGTGGGTTGTTCGTCCGGGT
TTTGTTAAAGGTAGCACCTGGGTTGAACCGCTGCCGGATGGTTTTCTGGG
TGAACGTGGTCGTATTGTTAAATGGGTTCCGCAGCAAGAAGTTCTGGCAC
ACGGCGCAATTGGTGCATTTTGGACCCATAGCGGTTGGAATAGCACCCTG
GAAAGCGTTTGTGAAGGTGTTCCGATGATTTTTAGCGATTTTGGTCTGGA
TCAGCCGCTGAATGCACGTTATATGAGTGATGTTCTGAAAGTGGGTGTGT
ATCTGGAAAATGGTTGGGAACGTGGTGAAATTGCAAATGCAATTCGTCGT
GTTATGGTGGATGAAGAAGGTGAATATATTCGTCAGAATGCCCGTGTICT
GAAACAGAAAGCAGATGTTAGCCTGATGAAAGGTGGTAGCAGCTATGAAA
GCCTGGAAAGTCTGGTTAGCTATATTAGCAGCCTGTAATAACTCGAG
[0307] After synthesis of the gene and subcloning into pET30A+
vector using NdeI and XhoI cloning sites, the UGT76G1_pET30a+
plasmid was introduced in E. coli B121(DE3) and E. coli EC100 by
electroporation. The obtained cells were grown in petri-dishes in
the presence of Kanamycin and suitable colonies were selected and
allowed to grow in liquid LB medium (erlenmeyer flasks). Glycerol
was added to the suspension as cryoprotectant and 400 .mu.L
aliquots were stored at -20.degree. C. and at -80.degree. C.
[0308] The storage aliquots of E. coli BL21(DE3) containing the
pET30A+_UGT76G1 plasmid were thawed and added to 30 mL of LBGKP
medium (20 g/L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50
mM Phosphate buffer pH 7.00; 2.5 g/L glucose and 50 mg/L of
Kanamycin). This culture was allowed to shake at 135 rpm at
30.degree. C. for 8 h.
[0309] The production medium contained 60 g/L of overnight express
instant TB medium (Novagen), 10 g/L of glycerol and 50 mg/L of
Kanamycin. The medium was allowed to stir at 20.degree. C. while
taking samples to measure the OD and pH. The cultures gave
significant growth and a good OD was obtained. After 40 h, the
cells were harvested by centrifugation and frozen to yield 12.7 g
of cell wet weight.
[0310] Lysis was performed by addition of Bugbuster Master mix
(Novagen) and the lysate was recovered by centrifugation and kept
frozen. Activity tests were performed with thawed lysate.
Example 2
[0311] In-vitro production of UGT76G1
[0312] The S30 T7 High Yield Protein expression system kit from
Promega was used. 4 .mu.g of UGT76G1_pET30a+ plasmid from E. coli
EC100 was mixed with 80 .mu.L of S30 premix plus and 72 .mu.L of
S30 T7 extract was added. Nuclease-free water was added in order to
obtain a total volume of 200 .mu.L and the resulting solution was
incubated for 2 h at 30.degree. C. 180 .mu.L was used in the
catalytic test reaction.
Example 3
[0313] In-Vitro Production of UGT91D2
[0314] NcoI and NdeI restriction sides were added to the original
nucleic sequence as described in Genbank accession no. ACE87855.1.
After codon optimization the following nucleic sequence was
obtained:
TABLE-US-00008 CCATGGCACATATGGCAACCAGCGATAGCATTGTTGATGATCGTAAACAG
CTGCATGTTGCAACCTTTCCGTGGCTGGCATTTGGTCATATTCTGCCGTA
TCTGCAGCTGAGCAAACTGATTGCAGAAAAAGGTCATAAAGTGAGCTTTC
TGAGCACCACCCGTAATATTCAGCGTCTGAGCAGCCATATTAGTCCGCTG
ATTAATGTTGTTCAGCTGACCCTGCCTCGTGTTCAAGAACTGCCGGAAGA
TGCCGAAGCAACCACCGATGTTCATCCGGAAGATATTCCGTATCTGAAAA
AAGCAAGTGATGGTCTGCAGCCGGAAGTTACCCGTTTTCTGGAACAGCAT
AGTCCGGATTGGATCATCTATGATTATACCCATTATTGGCTGCCGAGCAT
TGCAGCAAGCCTGGGTATTAGCCGTGCACATTTTAGCGTTACCACCCCGT
GGGCAATTGCATATATGGGTCCGAGCGCAGATGCAATGATTAATGGTAGT
GATGGTCGTACCACCGTTGAAGATCTGACCACCCCTCCGAAATGGTTTCC
GTTTCCGACCAAAGTTTGTTGGCGTAAACATGATCTGGCACGTCTGGTTC
CGTATAAAGCACCGGGTATTAGTGATGGTTATCGTATGGGTCTGGTTCTG
AAAGGTAGCGATTGTCTGCTGAGCAAATGCTATCATGAATTTGGCACCCA
GTGGCTGCCGCTGCTGGAAACCCTGCATCAGGTTCCGGTTGTTCCGGTGG
GTCTGCTGCCTCCGGAAGTTCCGGGTGATGAAAAAGATGAAACCTGGGTT
AGCATCAAAAAATGGCTGGATGGTAAACAGAAAGGTAGCGTGGTTTATGT
TGCACTGGGTAGCGAAGTTCTGGTTAGCCAGACCGAAGTTGTTGAACTGG
CACTGGGTCTGGAACTGAGCGGTCTGCCGTTTGTTTGGGCATATCGTAAA
CCGAAAGGTCCGGCAAAAAGCGATAGCGTTGAACTGCCGGATGGTTTTGT
TGAACGTACCCGTGATCGTGGTCTGGTTTGGACCAGCTGGGCACCTCAGC
TGCGTATTCTGAGCCATGAAAGCGTTTGTGGTTTTCTGACCCATTGTGGT
AGCGGTAGCATTGTGGAAGGTCTGATGTTTGGTCATCCGCTGATTATGCT
GCCGATTTTTGGTGATCAGCCGCTGAATGCACGTCTGCTGGAAGATAAAC
AGGTTGGTATTGAAATTCCGCGTAATGAAGAAGATGGTTGCCTGACCAAA
GAAAGCGTTGCACGTAGCCTGCGTAGCGTTGTTGTTGAAAAAGAAGGCGA
AATCTATAAAGCCAATGCACGTGAACTGAGCAAAATCTATAATGATACCA
AAGTGGAAAAAGAATATGTGAGCCAGTTCGTGGATTATCTGGAAAAAAAC
ACCCGTGCAGTTGCCATTGATCACGAAAGCTAATGACTCGAG
[0315] After synthesis of the gene and subcloning into pET30A+
vector using NcoI and XhoI cloning sites, the UGT91D2 pET30a+
plasmid was introduced into E. coli EC100 by electroporation. The
obtained cells were grown in the presence of Kanamycin and suitable
colonies were selected and allowed to grow in liquid LB medium
(erlenmeyer flasks). Glycerol was added to the suspension as
cryoprotectant and 400 .mu.L aliquots were stored at -20.degree. C.
and at -80.degree. C.
[0316] The S30 T7 High Yield Protein expression system kit from
Promega was used for the in-vitro synthesis of the protein.
[0317] 4 .mu.g of UGT91D2_pET30a+ plasmid was mixed with 80 .mu.L
of S30 premix plus and 72 .mu.L of S30 T7 extract was added.
Nuclease-free water was added in order to obtain a total volume of
200 .mu.L and the resulting solution was incubated for 2 h at
30.degree. C. 5 .mu.L was used for SDS-page analysis while the
remaining 45 .mu.L was used in the catalytic test reaction.
Example 4
[0318] Catalytic Reaction with In-Vivo Produced UGT76G1
[0319] The total volume of the reaction was 5.0 mL with the
following composition: 50 mM sodium phosphate buffer pH 7.2, 3 mM
MgCl.sub.2, 2.5 mM UDP-glucose, 0.5 mM Stevioside and 500 .mu.l, of
UGT76G1 thawed lysate. The reactions were run at 30.degree. C. on
an orbitary shaker at 135 rpm. For each sample, 460 .mu.L of the
reaction mixture was quenched with 40 .mu.L of 2N H.sub.2SO.sub.4
and 420 .mu.L of methanol/water (6/4). The samples were immediately
centrifuged and kept at 10.degree. C. before analysis by HPLC
(CAD). HPLC indicated almost complete conversion of stevioside to
rebaudioside A, as shown in FIG. 51.
Example 5
[0320] Catalytic Reaction with In-Vitro Produced UGT91D2
[0321] The total volume of the reaction was 0.5 mL with the
following composition: 50 mM sodium phosphate buffer pH 7.2, 3 mM
MgCl.sub.2, 3.8 mM UDP-glucose, 0.1 mM Rebaudioside A and 180 .mu.L
of in-vitro produced UGT91D2. The reactions were run at 30.degree.
C. on an orbitary shaker at 135 rpm. For each sample, 450 .mu.l of
reaction mixture was quenched with 45 .mu.L of 2N H.sub.2SO.sub.4
and 405 .mu.L of 60% MeOH. After centrifugation, the supernatant
was analyzed by HPLC (CAD). HPLC indicated a 4.7% conversion of
rebaudioside A to rebaudioside D after 120 h.
Example 6
[0322] Catalytic Reaction with In-Vitro Produced UGT76G1
[0323] The total volume of the reaction was 2 mL with the following
composition: 50 mM sodium phosphate buffer pH 7.2, 3 mM MgCl.sub.2,
3.8 mM UDP-glucose, 0.5 mM Rebaudioside D and 180 .mu.L of in-vitro
produced UGT76G1. The reactions were run at 30.degree. C. on an
orbitary shaker at 135 rpm. For each sample, 400 .mu.I, of reaction
mixture was quenched with 40 .mu.L of 2N H.sub.2SO.sub.4 and 360
.mu.L of 60% MeOH. After centrifugation, the supernatant was
analyzed by HPLC (CAD). HPLC indicated 80% conversion of
rebaudioside D to rebaudioside M after 120 h as shown in FIG.
52.
[0324] For examples 7 to 12, the following abbreviations were
used:
[0325] LBGKP medium: 20 g/L Luria Broth Lennox; 50 mM PIPES buffer
pH 7.00; 50 mM Phosphate buffer pH 7.00; 2.5 g/L glucose and 50
mg/L of Kanamycin or Ampicillin
[0326] LB medium: (20 g/L Luria Broth Lennox)
Example 7
[0327] Preparation and Activity of UGT76G1 Prepared by pET30a+
Plasmid and BL21 (DE3) Expression Strain
[0328] The pET30a+_UGT76G1 plasmid was transformed into BL21(DE3)
expression strain (Lucigen E. Cloni.RTM. EXPRESS Electrocompetent
Cells). The obtained cells were grown on LB Agar medium in
petri-dishes in the presence of Kanamycin. Suitable colonies were
selected and allowed to grow in liquid LBGKP medium containing
Kanamycin. Glycerol was added and 400 .mu.L aliquots were stored at
-20.degree. C. and at -80.degree. C.
[0329] A storage aliquot was thawed and added to 30 mL of LBGKP
medium. This culture was allowed to shake at 30.degree. C. for 8 h.
and subsequently used to inoculate 400 mL of production medium
containing 60 g/L of "Overnight express instant TB medium"
(Novagen, reference 71491-5), 10 g/L of glycerol and 50 mg/L of
Kanamycin. The medium was allowed to stir at 20.degree. C. while
taking samples to measure the OD (600 nm) and pH. After 40 h, the
cells were harvested by centrifugation and frozen. The obtained
cell wet weight was 10.58 g.
[0330] 3.24 g of obtained pellet was lysed by addition of 8.1 mL of
"Bugbuster Master mix" (Novagen, reference 71456) and 3.5 mL of
water. The lysate was recovered by centrifugation and kept
frozen.
Example 8
[0331] Preparation and Activity of UGT76G1 Prepared by pET30a+
Plasmid and Tuner (DE3) Expression Strain
[0332] The pET30a+_UGT76G1 plasmid was transformed into Tuner (DE3)
expression strain (Novagen Tune.TM. (DE3) Competent cells) by heat
shock treatment. The obtained cells were grown on LB Agar medium in
petri-dishes in the presence of Kanamycin. Suitable colonies were
selected and allowed to grow in liquid LBGKP medium containing
Kanamycin). Glycerol was added and 400 .mu.L aliquots were stored
at -20.degree. C. and at -80.degree. C.
[0333] A storage aliquot was thawed and added to 100 mL of LB
medium containing 50 mg/L of Kanamycin. This culture allowed to
shake at 30.degree. C. for 15 h. 4.4 mL of this culture was used to
inoculate 200 mL of production medium containing LB. This medium
was allowed to stir at 37.degree. C. until an OD (600 nm) of 0.9
was obtained, after which 400 .mu.L of a 100 mM IPTG solution was
added and the medium was allowed to stir at 30.degree. C. for 4 h.
The cells were harvested by centrifugation and frozen. The obtained
cell wet weight was 1.38 g.
[0334] The obtained pellet was lysed by addition of 4.9 mL of
"Bugbuster Master mix" (Novagen, reference 71456) and 2.1 mL of
water. The lysate was recovered by centrifugation and kept
frozen.
Example 9
[0335] Preparation and Activity of UGT76G1 Prepared by pMAL Plasmid
and BL21 Expression Strain
[0336] After subcloning the synthetic UGT76G1 gene into the pMAL
plasmid using NdeI and Sal1 cloning sites, the pMAL_UGT76G1 plasmid
was transformed into BL21 expression strain (New England Biolabs
BL21 Competent E. coli) by heat shock treatment. The obtained cells
were grown on LB Agar medium in petri-dishes in the presence of
Ampicillin. Suitable colonies were selected and allowed to grow in
liquid LBGKP medium containing Ampicillin). Glycerol was added and
400 .mu.L aliquots were stored at -20.degree. C. and at -80.degree.
C.
[0337] A storage aliquot was thawed and added to 30 mL of LBGKP
medium. This culture was allowed to shake at 30.degree. C. for 8 h.
and subsequently used to inoculate 400 mL of production medium
containing 60 g/L of "Overnight express instant TB medium"
(Novagen, reference 71491-5), 10 g/L of glycerol and 50 mg/L of
Ampicillin. The medium was allowed to stir at 20.degree. C. while
taking samples to measure the OD and pH. After 40 h, the cells were
harvested by centrifugation and frozen. The obtained cell wet
weight was 5.86 g.
[0338] 2.74 g of obtained pellet was lysed by addition of 9.6 mL of
"Bugbuster Master Mix" (Novagen, reference 71456) and 4.1 mL of
water. The lysate was recovered by centrifugation and kept
frozen.
Example 10
[0339] Preparation and Activity of UGT76G1 Prepared by pMAL Plasmid
and ArcticExpress Expression Strain
[0340] The pMAL_UGT76G1 plasmid was transformed into ArticExpress
expression strain (Agilent ArcticExpress competent cells) by heat
shock treatment. The obtained cells were grown on LB Agar medium in
petri-dishes in the presence of Ampicillin and Geneticin. Suitable
colonies were selected and allowed to grow in liquid LBGKP medium
containing of Ampicillin and Geneticin. Glycerol was added and 400
.mu.L aliquots were stored at -20.degree. C. and at -80.degree.
C.
[0341] A storage aliquot was thawed and added to 30 mL of LBGKP
medium (containing Ampicillin and Geneticin). This culture was
allowed to shake at 30.degree. C. for 8 h. and subsequently used to
inoculate 400 mL of production medium containing 60 g/L of
"Overnight express instant TB medium" (Novagen, reference 71491-5),
10 g/L of glycerol and 50 mg/L of Ampicillin. The medium was
allowed to stir at 12.degree. C. while taking samples to measure
the OD (600 nm) and pH. After 68 h, the cells were harvested by
centrifugation and frozen. The obtained cell wet weight was 8.96
g.
[0342] 2.47 g of the obtained pellet was lysed by addition of 8.73
mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 3.79 mL
of water. The lysate was recovered by centrifugation and kept
frozen.
Example 11
[0343] Preparation and Activity of UGT76G1 Prepared by pCOLDIII
Plasmid and ArcticExpress Expression Strain
[0344] After subcloning the synthetic UGT76G1 gene into the
pCOLDIII plasmid using NdeI and XhoI cloning sites, the
pCOLDIII_UGT76G1 plasmid was transformed into ArcticExpress
expression strain (Agilent ArcticExpress competent cells) by heat
shock treatment. The obtained cells were grown on LB Agar medium in
petri-dishes in the presence of Ampicillin and Geneticin. Suitable
colonies were selected and allowed to grow in liquid LBGKP medium
containing Ampicillin and Geneticin. Glycerol was added and 400
.mu.L aliquots were stored at -20.degree. C. and at -80.degree.
C.
[0345] A storage aliquot was thawed and added to 30 mL of LBGKP
medium (containing Ampicillin and Geneticin). This culture was
allowed to shake at 30.degree. C. for 8 h. and subsequently used to
inoculate 400 mL of production medium containing 60 g/L of
"Overnight express instant TB medium" (Novagen, reference 71491-5),
10 g/L of glycerol and 50 mg/L of Kanamycin. The medium was allowed
to stir at 12.degree. C. while taking samples to measure the OD
(600 nm) and pH. After 63 h, the cells were harvested by
centrifugation and frozen. The obtained cell wet weight was 6.54
g.
[0346] 2.81 g of the obtained pellet was lysed by addition of 9.8
mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 4.2 mL
of water. The lysate was recovered by centrifugation and kept
frozen.
Example 12
[0347] Preparation and Activity of UGT76G1 Prepared by pCOLDIII
Plasmid and Origami2 (DE3) Expression Strain
[0348] The pCOLDIII_UGT76G1 plasmid was transformed into Origami2
(DE3) expression strain (Novagen Origami.TM.2 (DE3) Competent
Cells) by heat shock treatment. The obtained cells were grown on LB
Agar medium in petri-dishes in the presence of Ampicillin. Suitable
colonies were selected and allowed to grow in liquid LBGKP medium
containing Ampicillin. Glycerol was added and 400 .mu.L aliquots
were stored at -20.degree. C. and at -80.degree. C.
[0349] A storage aliquot was thawed and added to 30 mL of LBGKP
medium (containing Ampicillin). This culture was allowed to shake
at 30.degree. C. for 8 h. and subsequently used to inoculate 400 mL
of production medium containing 60 g/L of "Overnight express
instant TB medium" (Novagen, reference 71491-5), 10 g/L of glycerol
and 50 mg/L of Kanamycin. The medium was allowed to stir at
12.degree. C. while taking samples to measure the OD (600 nm) and
pH. After 68 h, the cells were harvested by centrifugation and
frozen. The obtained cell wet weight was 2.53 g.
[0350] 1.71 g of the obtained pellet was lysed by addition of 6.0
mL of "Bugbuster Master mix" (Novagen, reference 71456) and 1.9 mL
of water. The lysate was recovered by centrifugation and kept
frozen.
Example 13
[0351] Determination of Activity
[0352] Activity tests were performed on a 5 mL scale with 500 .mu.L
of thawed lysate for the transformation of Stevioside to
Rebaudioside A and Rebaudioside D to Rebaudioside M using 0.5 mM of
substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl.sub.2 in 50 mM
Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed
by HPLC. The results for the different preparations of UGT76G1 are
summarized in the following table.
TABLE-US-00009 Transformation activity* Stevioside Rebaudioside
Exam- Expression to Rebaudio- D to Rebaudio- ple Plasmid strain
side A side M 7 pET30a+ BL21 (DE3) 29 U mL.sup.-1 0.31 U mL.sup.-1
8 pET30a+ Tuner (DE3) 33 U mL.sup.-1 0.40 U mL.sup.-1 9 pMAL BL21
20 U mL.sup.-1 0.15 U mL.sup.-1 10 pMAL ArcticExpress 15 U
mL.sup.-1 0.25 U mL.sup.-1 11 pCOLDIII ArcticExpress 15 U mL.sup.-1
0.11 U mL.sup.-1 12 pCOLDIII Origami2 (DE3) 37 U mL.sup.-1 0.20 U
mL.sup.-1 *Note The activities for the transformation of Stevioside
and Rebaudioside M are mentioned per mL of lysate. 1 U will
transform 1 .mu.mol of substance in 1 hour at 30.degree. C. and pH
7.2
Example 14
[0353] 50 mL Scale Reaction for the Transformation of Rebaudioside
D to Rebaudioside M
[0354] 5 mL of the lysate of Example 12 was used to transform
Rebaudioside D to Rebaudioside M on a 50 mL scale. The reaction
medium consisted of 50 mM Sodium Phosphate buffer pH 7.2, 3 mM of
MgCl.sub.2, 2.5 mM of UDP-Glucose and 0.5 mM of Rebaudioside D.
After allowing the reaction to be shaken at 30.degree. C. for 90 h.
50 mL of ethanol was added and the resulting mixture was allowed to
stir at -20.degree. C. for 1 h. After centrifugation at 5000 g for
10 min. the supernatant was purified via ultrafiltration (Vivaflow
MWCO 30000). 78 mL of permeate was obtained and the 9 mL of
retentate was diluted with 9 mL of ethanol and resubjected to
Ultrafiltration (Vivaflow MWCO 30000). Another 14 mL of filtrate
was obtained, which was combined with the first permeate. The
combined permeates were concentrated under reduced pressure at
30.degree. C. until 32 mL of a clear solution was obtained.
[0355] The HPLC trace of the product mixture is shown in FIG. 5.
HPLC was carried out on an Agilent 1200 series equipped with a
binary pump, auto sampler, and thermostat column compartment. The
method was isocratic, with a mobile phase composed of 70% water
(0.1% formic acid): 30% acetonitrile. The flow rate was 0.1
.mu.L/min. The column used was Phenomenex Prodigy 5.mu. ODS (3) 100
A; 250.times.2 mm. The column temperature was maintained at
40.degree. C. The injection volume was 20-40 .mu.l.
Example 15
[0356] Preparation of UGT91D2 Using pMAL Plasmid and BL21
Expression Strain
[0357] After subcloning the synthetic UGT91D2 gene into the pMAL
plasmid using NdeI and Sal1 cloning sites, the pMAL_UGT91D2 plasmid
was transformed into BL21 expression strain (New England Biolabs
BL21 Competent E. coli) by heat shock treatment. The obtained cells
were grown on LB Agar medium in petri-dishes in the presence of
Ampicillin. Suitable colonies were selected and allowed to grow in
liquid LBGKP medium containing Ampicillin). Glycerol was added and
400 .mu.L aliquots were stored at -20.degree. C. and at -80.degree.
C.
[0358] A storage aliquot was thawed and added to 30 mL of LBGKP
medium. This culture was allowed to shake at 30.degree. C. for 8 h.
and subsequently used to inoculate 400 mL of production medium
containing 60 g/L of "Overnight express instant TB medium"
(Novagen, reference 71491-5), 10 g/L of glycerol and 50 mg/L of
Ampicillin. The medium was allowed to stir at 20.degree. C. while
taking samples to measure the OD and pH. After 40 h, the cells were
harvested by centrifugation and frozen. The obtained cell wet
weight is 12.32 g.
[0359] 2.18 g of obtained pellet was lysed by addition of 7.7 mL of
"Bugbuster Master Mix" (Novagen, reference 71456) and 3.2 mL of
water. The lysate was recovered by centrifugation and used directly
for activity testing.
Example 16
[0360] Preparation of UGT91D2 Using pMAL Plasmid and ArcticExpress
Expression Strain
[0361] The pMAL_UGT91D2 plasmid was transformed into ArcticExpress
expression strain (Agilent ArcticExpress competent cells) by heat
shock treatment. The obtained cells were grown on LB Agar medium in
petri-dishes in the presence of Ampicillin and Geneticin. Suitable
colonies were selected and allowed to grow in liquid LBGKP medium
containing Ampicillin and Geneticin. Glycerol was added and 400
.mu.L aliquots were stored at -20.degree. C. and at -80.degree.
C.
[0362] A storage aliquot was thawed and added to 30 mL of LBGKP
medium (containing Ampicillin and Geneticin). This culture was
allowed to shake at 30.degree. C. for 8 h. and subsequently used to
inoculate 400 mL of production medium containing 60 g/L of
"Overnight express instant TB medium" (Novagen, reference 71491-5),
10 g/L of glycerol and 50 mg/L of Ampicillin. The medium was
allowed to stir at 20.degree. C. for 16 h. followed by another 50
h. at 12.degree. C. while taking samples to measure the OD (600 nm)
and pH. The cells were harvested by centrifugation and frozen. The
obtained cell wet weight is 15.77 g.
[0363] 2.57 g of the obtained pellet was lysed by addition of 9.0
mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 3.8 mL
of water. The lysate was recovered by centrifugation and used
directly for activity testing.
Example 17
[0364] Preparation of UGT91D2 Using pET30a+ Plasmid and Tuner (DE3)
Expression Strain
[0365] The pET30a+_UGT91D2 plasmid was transformed into Tuner (DE3)
expression strain (Novagen Tuner.TM. (DE3) Competent cells) by heat
shock treatment. The obtained cells were grown on LB Agar medium in
petri-dishes in the presence of Kanamycin. Suitable colonies were
selected and allowed to grow in liquid LBGKP medium (containing
Kanamycin). Glycerol was added and 400 .mu.L aliquots were stored
at -20.degree. C. and at -80.degree. C.
[0366] A storage aliquot was thawed and added to 100 mL of LB
medium containing 50 mg/L of Kanamycin. This culture allowed to
shake at 30.degree. C. for 15 h. 6.2 mL of this culture was used to
inoculate 500 mL of production medium containing LB. This medium
was allowed to stir at 37.degree. C. until an OD (600 nm) of 0.9
was obtained after which 500 .mu.L of a 100 mM IPTG solution was
added (IPTG concentration in medium is 100 .mu.M) and the medium
was allowed to stir at 30.degree. C. for 4 h, the cells were
harvested by centrifugation and frozen. The obtained cell wet
weight is 4.02 g.
[0367] 1.92 g of the obtained pellet was lysed by addition of 6.8
mL of "Bugbuster Master mix" (Novagen, reference 71456) and 2.8 mL
of water. The lysate was recovered by centrifugation and tested
directly for activity.
Example 18
[0368] Preparation of UGT91D2 Using pET30a+ Plasmid and
ArcticExpress Expression Strain
[0369] The pET30a+_UGT91D2 plasmid was transformed into
ArcticExpress (DE3) expression strain (Agilent ArcticExpress
competent cells) by heat shock treatment. The obtained cells were
grown on LB Agar medium in petri-dishes in the presence of
Kanamycin and Geneticin. Suitable colonies were selected and
allowed to grow in liquid LBGKP medium containing of Kanamycin and
Geneticin. Glycerol was added and 400 .mu.L aliquots were stored at
-20.degree. C. and at -80.degree. C.
[0370] A storage aliquot was thawed and added to 30 mL of LBGKP
medium (containing Kanamycin and Geneticin). This culture was
allowed to shake at 30.degree. C. for 8 h. and subsequently used to
inoculate 400 mL of production medium containing 60 g/L of
"Overnight express instant TB medium" (Novagen, reference 71491-5),
10 g/L of glycerol and 50 mg/L of Ampicillin. The medium was
allowed to stir at 20.degree. C. for 16h. followed by another 50 h.
at 12.degree. C. while taking samples to measure the OD (600 nm)
and pH. After 60 h, the cells were harvested by centrifugation and
frozen. The obtained cell wet weight is 16.07 g.
[0371] 3.24 g of the obtained pellet was lysed by addition of 11.4
mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 4.8 mL
of water. The lysate was recovered by centrifugation and used
directly for activity testing.
Example 19
[0372] Determination of Activity of In-Vivo Preparations of
UGT91D2
[0373] Activity tests were performed at 5 mL scale with 1000 .mu.L
of lysate for the transformation of Rubusoside to Stevioside using
0.5 mM of substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl.sub.2 in
50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and
analyzed by HPLC. The results for the different preparations of
UGT91D2 are summarized in the following table.
TABLE-US-00010 Exam- Transformation activity* ple Plasmid
Expression strain Rubusoside to Stevioside 15 pMAL BL21 9 mU
mL.sup.-1 16 pMAL ArcticExpress 60 mU mL.sup.-1 17 pET30a+ Tuner
(DE3) 28 mU mL.sup.-1 18 pET30a+ ArcticExpress (DE3) 21 mU
mL.sup.-1 *Note: The activities are mentioned per mL of lysate. 1 U
will transform 1 .mu.mol of substrate in 1 hour at 30.degree. C.
and pH 7.2
Example 20
[0374] Other Enzymes for Rebaudioside A to Rebaudioside D
Conversion
[0375] The following genes of UDP-glucosyltransferases were
identified from public databases, synthesized by DNA2.0 and
subsequently subcloned in pET30a+ vector.
TABLE-US-00011 Internal Conversion Microplate Position Gene Name
reference RebA to RebD C908201 A1 gi115454819_NP_001051010.1
S115N01 A1 Active C908201 G2 gi187373030_ACD03249.1 S115N01 G2
Active C908201 A7 gi460409128_XP_004249992.1 S115N05 A7 Active
C912666 E1 gi222619587_EEE55719.1 S115N06 E1 Active C912666 C2
gi297795735_XP_002865752.1 S115N06 C2 Active
[0376] The aminoacid sequences are as follows:
[0377] >gi|115454819|ref|NP_001051010.1|Os03g0702500 [Oryza
sativa Japonica Group]
TABLE-US-00012 MDDAHSSQSPLHVVIFPWLAFGHLLPCLDLAERLAARGHRVSFVSTPRNL
ARLPPVRPELAELVDLVALPLPRVDGLPDGAEATSDVPFDKFELHRKAFD
GLAAPFSAFLDTACAGGKRPDWVLADLMHHWVALASQERGVPCAMILPCS
AAVVASSAPPTESSADQREAIVRSMGTAAPSFEAKRATEEFATEGASGVS
IMTRYSLTLQRSKLVAMRSCPELEPGAFTILTRFYGKPVVPFGLLPPRPD
GARGVSKNGKHDAIMQWLDAQPAKSVVYVALGSEAPMSADLLRELAHGLD
LAGTRFLWAMRKPAGVDADSVLPAGFLGRTGERGLVTTRWAPQVSILAHA
AVCAFLTHCGWGSVVEGLQFGHPLIMLPILGDQGPNARILEGRKLGVAVP
RNDEDGSFDRGGVAGAVRAVVVEEEGKTFFANARKLQEIVADREREERCI
DEFVQHLTSWNELKNNSDGQYP
[0378] >gi|187373030|gb|ACD03249.1|UDP-glycosyltransferase
[Avena strigosa]
TABLE-US-00013 MAVKDEQQSPLHILLFPFLAPGHLIPIADMAALFASRGVRCTILTTPVNA
AIIRSAVDRANDAFRGSDCPAIDISVVPFPDVGLPPGVENGNALTSPADR
LKFFQAVAELREPFDRFLADNIAPDAVVSDSFFHWSTDAAAEHGVPRLGF
LGSSMFAGSCNESTLHNNPLETAADDPDALVSLPGLPHRVELRRSQMMDP
KKRPDHWALLESVNAADQKSFGEVFNSFHELEPDYVEHYQTTLGRRTWLV
GPVALASKDMAGRGSTSARSPDADSCLRWLDTKQPGSVVYVSFGTLIRFS
PAELHELARGLDLSGKNFVWVLGRAGPDSSEWMPQGFADLITPRGDRGFI
IRGWAPQMLILNHRALGGFVTHCGWNSTLESVSAGVPMVTWPRFADQFQN
EKLIVEVLKVGVSIGAKDYGSGIENHDVIRGEVIAESIGKLMGSSEESDA
IQRKAKDLGAEARSAVENGGSSYNDVGRLMDELMARRSSVKVGEDIIPTN DGL
[0379] >gi|460409128|ref|XP_004249992.1| PREDICTED:
cyanidin-3-O-glucoside 2-O-glucuronosyltransferase-like [Solanum
lycopersicum]
TABLE-US-00014 MSPKLHKELFFHSLYKKTRSNHTMATLKVLMFPFLAYGHISPYLNVAKKL
ADRGFLIYFCSTPINLKSTIEKIPEKYADSIHLIELHLPELPQLPPHYHT
TNGLPPNLNQVLQKALKMSKPNFSKILQNLKPDLVIYDILQRWAKHVANE
QNIPAVKLLTSGAAVFSYFFNVLKKPGVEFPFPGIYLRKIEQVRLSEMMS
KSDKEKELEDDDDDDDLLVDGNMQIMLMSTSRTIEAKYIDFCTALTNWKV
VPVGPPVQDLITNDVDDMELIDWLGTKDENSTVFVSFGSEYFLSKEDMEE
VAFALELSNVNFIWVARFPKGEERNLEDALPKGFLERIGERGRVLDKFAP
QPRILNHPSTGGFISHCGWNSAMESIDFGVPIIAMPMHLDQPMNARLIVE
LGVAVEIVRDDDGKIHRGEIAETLKGVITGKTGEKLRAKVRDISKNLKTI
RDEEMDAAAEELIQLCRNGN
[0380] >gi|222619587|gb|EEE55719.1| hypothetical protein
OsJ_04191 [Oryza sativa Japonica Group]
TABLE-US-00015 MHVVMLPWLAFGHILPFAEFAKRVARQGHRVTLFSTPRNTRRLIDVPPSL
AGRIRVVDIPLPRVEHLPEHAEATIDLPSNDLRPYLRRAYDEAFSRELSR
LLQETGPSRPDWVLADYAAYWAPAAASRHGVPCAFLSLFGAAALCFFGPA
ETLQGRGPYAKTEPAHLTAVPEYVPFPTTVAFRGNEARELFKPSLIPDES
GVSESYRFSQSIEGCQLVAVRSNQEFEPEWLELLGELYQKPVIPIGMFPP
PPPQDVAGHEETLRWLDRQEPNSVVYAAFGSEVKLTAEQLQRIALGLEAS
ELPFIWAFRAPPDAGDGDGLPGGFKERVNGRGVVCRGWVPQVKFLAHASV
GGFLTHAGWNSIAEGLANGVRLVLLPLMFEQGLNARQLAEKKVAVEVARD
EDDGSFAANDIVDALRRVMVGEEGDEFGVKVKELAKVFGDDEVNDRYVRD
FLKCLSEYKMQRQG
[0381] >gi|297795735|ref|XP_002865752.1|
UDP-glucoronosyl/UDP-glucosyl transferase family protein
[Arabidopsis lyrata subsp. lyrata]
TABLE-US-00016 MDDKKEEVMHIAMFPWLAMGHLLPFLRLSKLLAQKGHKISFISTPRNILR
LPKLPSNLSSSITFVSFPLPSISGLPPSSESSMDVPYNKQQSLKAAFDLL
QPPLTEFLRLSSPDWIIYDYASHWLPSIAKELGISKAFFSLFNAATLCFM
GPSSSLIEESRSTPEDFTVVPPWVPFKSTIVFRYHEVSRYVEKTDEDVTG
VSDSVRFGYTIDGSDAVFVRSCPEFEPEWFSLLQDLYRKPVFPIGFLPPV
IEDDDDDTTWVRIKEWLDKQRVNSVVYVSLGTEASLRREELTELALGLEK
SETPFFWVLRNEPQIPDGFEERVKGRGMVHVGWVPQVKILSHESVGGFLT
HCGWNSVVEGIGFGKVPIFLPVLNEQGLNTRLLQGKGLGVEVLRDERDGS
FGSDSVADSVRLVMIDDAGEEIREKVKLMKGLFGNMDENIRYVDELVGFM
RNDESSQLKEEEEEDDCSDDQSSEVSSETDEKELNLDLKEEKRRISVYKS
LSSEFDDYVANEKMG
[0382] The tested plasmids were received in a microtiterplate
containing a plasmid as freeze-dried solid in each separate
well.
[0383] Suspension of Plasmids.
[0384] To each well was added 24 .mu.L of ultra-pure sterile water
and the microtiter plate was shaken for 30 minutes at Room
Temperature. Subsequently, the plate was incubated at 4.degree. C.
for 1 hour. The content of each well were further mixed by
pipetting up and down. The plasmid quantification was performed by
Qubit2.0 analysis using 1 .mu.L of suspension. Determined
quantities of plasmids were:
TABLE-US-00017 Internal Microtiter plate Position reference
[Plasmid] ng/.mu.L C908201 A1 S115N01 A1 32.8 C908201 G2 S115N01 G2
41.0 C908201 A7 S115N05 A7 56.6 C912666 E1 S115N06 E1 64.0 C912666
C2 S115N06 C2 31.4
[0385] Transformation of Competent Cells with Plasmids.
[0386] Aliquots of chemically competent EC100 cells were taken from
freezer at -80.degree. C. and stored on ice. The cells were allowed
to thaw on ice for 10 minutes. 10 .mu.L of a dilution of above
described plasmid solution was added to a sterile microtube of 1.5
mL (in order to transform each cell with 50 pg of DNA) and stored
on ice. 100 .mu.L of chemically competent cells was added to each
microtube. After incubation of the chemically competent cells
plasmid mixtures on ice for 20 min a thermal shock of 30 seconds at
42.degree. C. was performed.
[0387] Further incubation was performed on ice for 2 minutes. To
each microtube 300 .mu.L of SOC medium was added and the resulting
mixture was transferred to a sterile 15 mL tube. After incubate for
1 hour at 37.degree. C. while shaking at 135 rpm, the mixture is
spread on solid Luria Broth medium containing Kanamycin 50
.mu.g/mL. The petri-dishes are allowed to incubate for 16 hours at
37.degree. C.
[0388] Preparation of Stock Solutions in Glycerol and Purification
of Plasmids.
[0389] To a 50 mL sterile Falcon Tube 10 mL of Luria Broth medium
containing 50 .mu.g/mL of Kanamycin was added. The medium was
seeded with an isolated colony from the above described Petri dish
and the cultures were allowed to incubate for 16 hours at
37.degree. C. while shaking at 135 rpm.
[0390] To sterile microtube of 1.5 mL containing 300 .mu.L of a 60%
sterile glycerol solution, 600 .mu.L of the culture was added. The
stock solution was stored at -80.degree. C.
[0391] The remainder of the culture was centrifuged at 5,525 g for
10 minutes at 10.degree. C. and after removal of the supernatant,
the pellet was stored on ice. The produced plasmids were purified
according to the Qiagen Qiaprep Spin Miniprep kit (ref: 27106) and
the plasmid yield was measured at 260 nm. The plasmid solution was
stored at 4.degree. C. Plasmid quantities were determined as
follows:
TABLE-US-00018 Microtiter Internal plate Position reference of test
[Plasmid] ng/.mu.L C908201 A1 S115N01 A1 115.7 C908201 G2 S115N01
G2 120.4 C908201 A7 S115N05 A7 293.8 C912666 E1 S115N06 E1 126.1
C912666 C2 S115N06 C2 98.8
[0392] In-Vitro Expression of Enzymes.
[0393] 18 .mu.L of plasmid solution (containing approximately 1.5
.mu.g of plasmid) was used for in-vitro expression according to the
Promega S30 T7 High-Yield Protein Expression System (ref: L1110)
kit. The expression medium was produced as follows:
TABLE-US-00019 S30 Premix Plus T7 S30 Extract Total Trials 30 .mu.L
27 .mu.L 57 .mu.L reference 20 .mu.L 18 .mu.L 38 .mu.L
[0394] The prepared expression medium mix was added to the plasmid
solution and the solution was allowed to incubate at 30.degree. C.
for 3 hours while mixing the mixture every 45 minutes. 5 .mu.L of
the mixture was frozen whereas the remainder was used for the
catalytic test for the conversion of Rebaudioside A to Rebaudioside
D.
[0395] Catalytic Test for Transformation of Rebaudioside A to
Rebaudioside D.
[0396] 430 .mu.L of a reaction mixture containing 0.5 mM
Rebaudioside A, 3 mM MgCl.sub.2, 50 mM phosphate buffer (pH7.2) and
2.5 mM UDP-glucose was added to a 1.5 mL sterile microtube. 52
.mu.l of the enzyme expression medium was added and the resulting
mixture was allowed to react at 30.degree. C. for 24 hours. 125
.mu.L samples were taken after 2 hours, 16 hours and 24 hours and
added to a 115 .mu.L of 60% methanol and 10 .mu.L of 2 N
H.sub.2SO.sub.4. The quenched sample was centrifuged at 18,000 g
for 2 minutes at RT. 200 .mu.L was transferred to an HPLC vial and
analyzed.
[0397] HPLC Analysis The HPLC assay was performed as follows:
[0398] Apparatus
TABLE-US-00020 Equipment Supplier Reference Lot# Elite Hitachi
L-2130 NA Photodiode Array Hitachi L-2455 NA Corona CAD detector
ESA 70-6186A CO-2044 Injector 100 .mu.L Hitachi NA Column Synergy 4
u Hydro- Phenomenex 00G-4375-E0 588582-12 RP 80A (250 .times. 4.60
mm)
[0399] Instrument Conditions
TABLE-US-00021 Column Temperature 55.degree. C. Detection UV 205
nm; bw 400 nm CAD detection Analysis duration 15 min Injected
volume 10 .mu.L Flow rate 1 mL/min
[0400] Mobile Phase Gradient Program
TABLE-US-00022 Time (min) % Water containing 0.04% acetic acid %
methanol 0 40 60 8 25 75 10 25 75 11 40 60 15 40 60
[0401] The HPLC assay results are provided below and shown in FIGS.
53a-e:
TABLE-US-00023 Steviol glycoside conversion Internal in reaction
mixture (% area) reference Reb D Reb UNK Reb A S115N01 A1 2.1 ND
96.7 S115N01 G2 0.6 ND 99.4 S115N05 A7 22.4 23.3 46.7 S115N06 E1
0.14 7.0 92.8 S115N06 C2 0.28 3.9 95.8
[0402] The enzyme S115N05 A7 had the highest activity for Reb A to
Reb D conversion (ca. 22.4%)
[0403] At least three enzymes produced a significant amount of an
unknown glycoside (marked as Reb UNK; later identified as reb D2)
along with reb D.
Example 21
[0404] Activity of In-Vitro Produced EUGT11
[0405] EUGT11 gene as was described in the Patent application
WO/2013/022989A2 was synthesized by DNA2.0 and subsequently
subcloned in pET30a+ vector.
TABLE-US-00024 Conversion Micro- Posi- GI Internal RebA to plate
tion number Version reference RebD C912666 G4 41469452 AAS07253.1
S115N08 G4 Active
[0406] The amino-acid sequence is as follows:
[0407] >gi|41469452|gb|AAS07253.1| putative UDP-glucoronosyl and
UDP-glucosyl transferase [Oryza sativa Japonica Group] EUGT11
enzyme from patent application WO/2013/022989A2
TABLE-US-00025 MHVVICPLLAFGHLLPCLDLAQRLACGHRVSFVSTPRNISRLPPVRPSLA
PLVSFVALPLPRVEGLPNGAESTHNVPHDRPDMVELHLRAFDGLAAPFSE
FLGTACADWVMPTSSAPRQTLSSNIHRNSSRPGTPAPSGRLLCPITPHSN
TLERAAEKLVRSSRQNARARSLLAFTSPPLPYRDVFRSLLGLQMGRKQLN
IAHETNGRRTGTLPLNLCRWMWKQRRCGKLRPSDVEFNTSRSNEAISPIG
ASLVNLQSIQSPNPRAVLPIASSGVRAVFIGRARTSTPTPPHAKPARSAA
PRAHRPPSSVMDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLAS
RGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTND
VPHDRPDMVELHRRAFDGLAAPFSEFLGTACADWVIVDVFHHWAAAAALE
HKVPCAMMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTFEVAR
MKLIRTKGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLLSTLRGK
PITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEK
VHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWV
PQMSILAHAAVGAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIE
AKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIV
ADMACHERYIDGFIQQLRSYKD
[0408] The tested plasmid was received in a microtiterplate
containing a plasmid as freeze-dried solid in a separate well.
[0409] Suspension of Plasmid
[0410] To the well was added 24 .mu.L of ultra-pure sterile water
and the microtiter plate was shaken for 30 minutes at Room
Temperature. Subsequently, the plate was incubated at 4.degree. C.
for 1 hour. The content of the well was further mixed by pipetting
up and down. The plasmid quantification was performed by Qubit2.0
analysis using 1 .mu.L of suspension. Plasmid quantity was
determined as follows:
TABLE-US-00026 Microtiter Internal plate Position reference of test
[Plasmid] ng/.mu.L C912666 G4 S115N08 G4 19.2
[0411] Transformation of Competent Cells with Plasmid.
[0412] An aliquot of chemically competent EC100 cells was taken
from freezer at -80.degree. C. and stored on ice. The cells were
allowed to thaw on ice for 10 minutes. 10 .mu.L of a dilution of
above described plasmid solution was added to a sterile microtube
of 1.5 mL (in order to transform each cell with 50 pg of DNA) and
stored on ice. 100 .mu.L of chemically competent cells was added to
the microtube. After incubation of the chemically competent
cells/plasmid mixture on ice for 20 min a thermal shock of 30
seconds at 42.degree. C. was performed.
[0413] Further incubation was performed on ice for 2 minutes. To
the microtube 300 .mu.L of SOC medium was added and the resulting
mixture was transferred to a sterile 15 mL tube. After incubate for
1 hour at 37.degree. C. while shaking at 135 rpm, the mixture is
spread on solid Luria Broth medium containing Kanamycin 50
.mu.g/mL. The Petri dish is allowed to incubate for 16 hours at
37.degree. C.
[0414] Preparation of Stock Solutions in Glycerol and Purification
of Plasmid.
[0415] To a 50 mL sterile Falcon Tube 10 mL of Luria Broth medium
containing 50 .mu.g/mL of Kanamycin was added. The medium was
seeded with an isolated colony from the above described Petri dish
and the cultures were allowed to incubate for 16 hours at
37.degree. C. while shaking at 135 rpm.
[0416] To sterile microtube of 1.5 mL containing 300 .mu.L of a 60%
sterile glycerol solution, 600 .mu.L of the culture was added. The
stock solution was stored at -80.degree. C.
[0417] The remainder of the culture was centrifuged at 5,525 g for
10 minutes at 10.degree. C. and after removal of the supernatant,
the pellet was stored on ice. The produced plasmids were purified
according to the Qiagen Qiaprep Spin Miniprep kit (ref: 27106) and
the plasmid yield was measured at 260 nm. The plasmid solution was
stored at 4.degree. C. Plasmid quantity was determined as
follows:
TABLE-US-00027 Microtiter Internal plate Position reference of test
[Plasmid] ng/.mu.L C912666 G4 S115N08 G4 38.4
[0418] In-Vitro Expression of EUGT11.
[0419] 18 .mu.L of a diluted plasmid solution (containing
approximately 1.5 .mu.g of plasmid) was used for in-vitro
expression according to the Promega S30 T7 High-Yield Protein
Expression System (ref: L1110) kit. The expression medium was
produced as follows:
TABLE-US-00028 S30 Premix Plus T7 S30 Extract DNA template Total
Trials 30 .mu.L 27 .mu.L 18 .mu.L (~1.5 .mu.g) 75 .mu.L reference
20 .mu.L 18 .mu.L 12 .mu.L (~1.0 .mu.g) 50 .mu.L
[0420] The prepared expression medium mix was added to the plasmid
solution and the solution was allowed to incubate at 30.degree. C.
for 3 hours while mixing the mixture every 45 minutes. 5 .mu.L of
the mixture was frozen whereas the remainder was used for the
catalytic test for the conversion of Rebaudioside A to Rebaudioside
D.
[0421] Catalytic Test for Transformation of Rebaudioside A to
Rebaudioside D.
[0422] 430 .mu.L of a reaction mixture containing 0.5 mM
Rebaudioside A, 3 mM MgCl.sub.2, 50 mM phosphate buffer (pH7.2) and
2.5 mM UDP-glucose was added to a 1.5 mL sterile microtube. 52
.mu.L of the enzyme expression medium was added and the resulting
mixture was allowed to react at 30.degree. C. for 24 hours. 125
.mu.L samples were taken after 2 hours, 16 hours and 24 hours and
added to a 115 .mu.L of 60% methanol and 10 .mu.L of 2 N
H.sub.2SO.sub.4. The quenched sample was centrifuged at 18,000 g
for 2 minutes at RT. 200 pt was transferred to HPLC vial and
analyzed.
[0423] HPLC Analysis.
[0424] The HPLC assay was performed as described in EXAMPLE 20.
[0425] The HPLC assay results are shown in FIG. 54.
Example 22
[0426] In-Vivo Production of Enzymes
[0427] The enzymes described in EXAMPLE 20 were produced in
vivo.
[0428] The pET30A+ vector containing the gene corresponding to the
enzyme was introduced in E. coli BL21(DE3) by heat shock. The
obtained cells were grown in Petri dishes in the presence of
Kanamycin and suitable colonies were selected and allowed to grow
in liquid LB medium (Erlenmeyer flasks). Glycerol was added to the
suspension as cryoprotector and 400 .mu.L aliquots were stored at
-20.degree. C. and at -80.degree. C.
[0429] The storage aliquots of E. coli BL21(DE3) containing the
pET30A+_UGT plasmids were thawed and added to 30 mL of LBGKP medium
(20 g/L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM
Phosphate buffer pH 7.00; 2.5 g/L glucose and 50 mg/L of
Kanamycine). This culture was allowed to shake at 135 rpm at
30.degree. C. for 8 hrs.
[0430] The production medium contained 60 g/L of overnight express
instant TB medium (Novagen), 10 g/L of glycerol and 50 mg/L of
Kanamycine. The preculture was added to 400 mL of this medium and
the solution was allowed to stir at 20.degree. C. while taking
samples to measure the OD and pH. The cultures gave significant
growth and a good OD was obtained. After 40 hrs, the cells were
harvested by centrifugation and frozen. The following yields of
cell wet weights (CWW) are mentioned below.
TABLE-US-00029 GI number Version CWW 115454819 NP_001051010.1 9.2 g
187373030 ACD03249.1 7.4 g 460409128 XP_004249992.1 6.8 g 222619587
EEE55719.1 7.5 g 297795735 XP_002865752.1 8.8 g
[0431] Lysis was performed by addition of Bugbuster Master mix
(Novagen) and the lysate was recovered by centrifugation and used
fresh.
[0432] Determination of Activity.
[0433] Activity tests were performed at 5 mL scale with 1,000 .mu.L
of thawed lysate for the transformation of Rebaudioside A using 0.5
mM of substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl.sub.2 in 50 mM
Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed
by HPLC.
[0434] HPLC Analysis.
[0435] The HPLC assay was performed as described in EXAMPLE 20.
[0436] The results for the different enzymes are provided below and
shown in FIGS. 55a-e.
TABLE-US-00030 Conversion Reb D GI Number Version after 45 hrs.
selectivity 15454819 NP_001051010.1 1.1% 100% 87373030 ACD03249.1
0.8% 100% 60409128 XP_004249992.1 62.1% 43.6% 22619587 EEE55719.1
2.9% Reb D Not detected 97795735 XP_002865752.1 0.0% Reb D Not
detected indicates data missing or illegible when filed
Example 23
[0437] Identification of Glycosides
[0438] The reaction mixtures representing GI No. 460409128,
particularly the sample "12400 S115N05A7 T24h 130627ABA" of EXAMPLE
20 (hereinafter S115N05A7), and the sample "12400 S129N04 T45h
130712ABA" of EXAMPLE 22 (hereinafter S129N04) were additionally
assayed by LC-MS to identify the unknown glycosides. An Agilent
1200 series HPLC system, equipped with binary pump (G1312B),
autosampler (G1367D), thermostatted column compartment (G1316B),
DAD detector (G1315C), connected with Agilent 6110A MSD, and
interfaced with "LC/MSD Chemstation" software, was used.
[0439] Instrument Conditions
TABLE-US-00031 Column Phenomenex Kinetex 2.6u C18 100 A, 4.6 mm
.times. 150 mm, 2.6 .mu.m Column Temperature 55.degree. C.
Detection DAD at 210 nm bw 360 nm MSD (Scan and SIM mode) Mode:
ES-API, Negative Polarity Drying gas flow: 13.0 L/min Nebulizer
pressure: 30 psig Drying gas temperature: 270.degree. C. Analysis
duration 25 min Injected volume 2 .mu.L Flow rate 1 mL/min
[0440] Mobile Phase Gradient Program
TABLE-US-00032 Time (min) A (%): Formic acid 0.1% B (%):
Acetonitrile 0 75 25 8.5 75 25 10.0 71 29 16.5 70 30
[0441] The compound observed on LCMS system at 3.5 min, corresponds
to compound "Unknown@4.508" in sample "S115N05A7" (EXAMPLE 20), and
compound "Unknown@RT4.526" in sample "S129N04" (EXAMPLE 22). The
LCMS data suggests that this compound has six glucosidic residues
(C.sub.56H.sub.90O.sub.33) in its structure, and was found to be an
isomer form of reb M, namely reb M2 (see Example 40 for
discussion).
[0442] Whereas the compound observed on LCMS system at 7.6 min,
corresponds with compound "reb UNK" in sample "S115N05A7" (EXAMPLE
20), and compound "reb UNK" in sample "S129N04" (EXAMPLE 22), The
LCMS data suggests that "reb UNK" has five glucosidic residues
(C.sub.50H.sub.80O.sub.28) in its structure, and was found to be an
isomer form of reb D, namely reb D2 (see Example 39 for
discussion). The ratio of these compounds and the LCMS
chromatograms are provided below.
TABLE-US-00033 Steviol glycoside conversion in reaction mixture (%
area) Sample Unknown@RT3.5 Reb D Reb UNK Reb A S115N05A7 6.47 20.35
19.93 53.24 S129N04 6.05 23.73 21.22 49.00
Example 24
[0443] Identification of Glycosides
[0444] The reaction mixture representing GI No. 460409128,
particularly the sample "12400 S129N04 T45h 130712ABA" of EXAMPLE
22 (hereinafter S129N04) were additionally assayed by LC-MS along
with Stevia rebaudiana Bertoni leaf extract "MLD1" produced by
PureCircle Sdn Bhd (Malaysia) to determine the occurrence of
S129N04 glycosides in nature.
[0445] The assays in FIGS. 57a-b show that the compound observed on
LCMS system at 3.5 min, in EXAMPLE 23 (C.sub.56H.sub.90O.sub.33;
later confirmed as reb M2), and the compound observed on LCMS
system at 7.6 min, in EXAMPLE 23 (C.sub.50H.sub.80O.sub.28; reb
UNK; later confirmed as reb D2) occur in the extract of Stevia
rebaudiana Bertoni plant.
Example 25
[0446] Conversion of Rebaudioside E to Rebaudioside D
[0447] The total volume of the reaction was 5.0 mL with the
following composition: 100 mM potassium phosphate buffer pH 7.5, 3
mM MgCl.sub.2, 2.5 mM UDP-glucose, 0.5 mM Rebaudioside E and 500
.mu.L of UGT76G1 thawed lysate (UGT76G1 gene was cloned in pET30a+
vector and expressed in E. coli BL21 (DE3)). The reactions were run
at 30.degree. C. on an orbitary shaker at 135 rpm. For sampling 300
.mu.L of the reaction mixture was quenched with 30 .mu.L of 2N
H.sub.2SO.sub.4 and 270 .mu.L of methanol/water (6/4). The samples
were immediately centrifuged and kept at 10.degree. C. before
analysis by HPLC (CAD detection). The reaction profile shown in
FIG. 58 was obtained corresponding to a complete conversion of
Rebaudioside E to Rebaudioside D.
Example 26
[0448] Directed Evolution of UGT76G1 for the Conversion of
Rebaudioside D to Rebaudioside M
[0449] Starting from the amino acid sequence of UGT76G1, as is
described in Genbank (AAR06912.1), different mutations at various
amino acid positions were identified that could alter the activity
of the enzyme for the transformation of Rebaudioside D (Reb to
Rebaudioside M (Reb M). This list of mutations, designed by DNA2.0
ProteinGPS.TM. strategy, was subsequently used to synthesize 96
variant genes that contained 3, 4 or 5 of these mutations that were
codon-optimized for expression in E. coli. The genes were subcloned
in the pET30a+ plasmid and used for transformation of E. coli BL21
(DE3) chemically competent cells. The obtained cells were grown in
Petri-dishes on solid LB medium in the presence of Kanamycin.
Suitable colonies were selected and allowed to grow in liquid LB
medium in tubes. Glycerol was added to the suspension as
cryoprotectant and 400 .mu.L aliquots were stored at -20.degree. C.
and at -80.degree. C.
[0450] These storage aliquots of E. coli BL21(DE3) containing the
pET30a+_UGT76G1var plasmids were thawed and added to LBGKP medium
(20 g/L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM
Phosphate buffer pH 7.00; 2.5 g/L glucose and 50 mg/L of
Kanamycine). This culture was allowed to shake in a 96 microtiter
plate at 135 rpm at 30.degree. C. for 8 h.
[0451] 3.95 mL of production medium containing 60 g/L of Overnight
Express.TM. Instant TB medium (Novagen.RTM.), 10 g/L of glycerol
and 50 mg/L of Kanamycin was inoculated with 50 .mu.L of above
described culture. In a 48 deepwell plate the resulting culture was
allowed to stir at 20.degree. C. The cultures gave significant
growth and a good OD (600 nm; 1 cm) was obtained. After 44 h, the
cells were harvested by centrifugation and frozen.
[0452] Lysis was performed by addition of Bugbuster.RTM. Master mix
(Novagen.RTM.) to the thawed cells and the lysate was recovered by
centrifugation. Activity tests were performed with 100 .mu.L of
fresh lysate that was added to a solution of Rebaudioside D (final
concentration 0.5 mM), MgCl.sub.2 (final concentration 3 mM) and
UDP-Glucose (final concentration 2.5 mM) in 50 mM phosphate buffer
pH 7.2.
[0453] The reaction was allowed to run at 30.degree. C. and samples
were taken after 2, 4, 7 and 24 h. to determine conversion and
initial rate by HPLC (CAD detection) using the analytical method
that was described above for the transformation of Rebaudioside D
to Rebaudioside M. The results are depicted in the following
table.
TABLE-US-00034 conversion Reb D initial rate to Reb M after 24 h
(Reb M Clone Mutations* (%) area/min) UGT76G1var1 E224A_F314S_R334K
51.8 5.5E+07 UGT76G1var2 S274G_T284I_L379G 49.3 4.7E+07 UGT76G1var3
I295T_S357C_V366I 9.6 1.6E+06 UGT76G1var4 E224D_E231A_F265I 14.7
8.6E+06 UGT76G1var5 F22Y_I373L_P382M 3.5 2.3E+06 UGT76G1var6
Q266S_S357N_I373L 0.5 1.8E+06 UGT76G1var7 F22L_I43V_A239V 0.2
-6.0E+04 UGT76G1var8 E224A_Q266S_Q342E 0.5 2.3E+04 UGT76G1var9
E231A_D301N_G348P 52.0 4.9E+07 UGT76G1var10 A33G_L246F_Q342E 0.3
-7.7E+02 UGT76G1var11 F22L_A33G_V310I 0.4 3.8E+04 UGT76G1var12
L243P_K303G_A352G 0.5 8.7E+04 UGT76G1var13 L243A_S357C_A385T 0.2
-3.3E+04 UGT76G1var14 A239I_F265I_V396F 5.3 1.5E+06 UGT76G1var15
F41L_L246F_Q425E 5.6 1.5E+06 UGT76G1var16 F265I_P272A_I335V 18.6
5.8E+06 UGT76G1var17 F265L_Q266E_Q342K 0.7 7.2E+05 UGT76G1var18
L243P_S274G_N409R 1.9 5.0E+05 UGT76G1var19 E224D_E229A_Q432E 10.5
5.5E+06 UGT76G1var20 S375M_K393G_Y397E 1.8 1.9E+06 UGT76G1var21
A239V_V300A_K303G 41.9 3.3E+07 UGT76G1var22 E231A_V310I_R334K 34.4
2.4E+07 UGT76G1var23 T263S_G348P_A352G 47.8 4.1E+07 UGT76G1var24
A239I_P272A_Q425E 31.0 2.1E+07 UGT76G1var25 T284L_Q342K_Y397Q 0.9
6.3E+04 UGT76G1var26 S241I_F265L_F377C 1.8 7.5E+05 UGT76G1var27
A239I_L379A_V394I 29.0 1.5E+07 UGT76G1var28 L243A_S274G_P382M 6.1
2.4E+06 UGT76G1var29 F22Y_V279I_N409R 41.0 2.9E+07 UGT76G1var30
I43V_E224A_S241I 13.6 5.6E+06 UGT76G1var31 E224D_L243P_V300A 0.4
2.4E+05 UGT76G1var32 A239V_L243A_S375M 0.0 -4.4E+04 UGT76G1var33
A33G_R334H_Y397Q 1.0 7.5E+06 UGT76G1var34 I43V_T284I_I295T 3.4
1.5E+06 UGT76G1var35 T284L_F314S_S357N 0.5 1.8E+05 UGT76G1var36
F265L_L379A_V396F 20.0 8.8E+06 UGT76G1var37 E229A_L379G_I407V 39.1
2.8E+07 UGT76G1var38 F41L_I295M_F377C 8.2 3.7E+06 UGT76G1var39
F22Y_F41L_V366I 7.2 3.3E+06 UGT76G1var40 T263S_Q266E_S375R 47.6
3.3E+07 UGT76G1var41 L246F_A385T_K393G 0.8 1.4E+06 UGT76G1var42
T263S_Q266S_R334H 34.6 2.2E+07 UGT76G1var43 S241I_P272A_V279I 19.9
9.4E+06 UGT76G1var44 I335V_S375R_I407V 35.3 2.3E+07 UGT76G1var45
V279I_D301N_S389E 38.6 2.3E+07 UGT76G1var46 F22L_Q266E_I295M 0.6
9.8E+05 UGT76G1var47 E229A_T284I_S389E 4.8 2.7E+06 UGT76G1var48
V394I_Y397E_Q432E 47.6 3.8E+07 UGT76G1var49 F41L_Q266E_T284I_Y397Q
2.6 1.1E+06 UGT76G1var50 F22Y_V310I_S375M_F377C 1.9 7.9E+05
UGT76G1var51 K303G_S357C_S389E_V396F 18.7 9.5E+06 UGT76G1var52
D301N_I373L_F377C_I407V 12.9 4.6E+06 UGT76G1var53
R334K_A352G_P382M_S389E 9.3 4.1E+06 UGT76G1var54
E229A_T284L_R334K_Q342E 0.7 4.3E+05 UGT76G1var55
I295M_Q342E_V366I_N409R 1.0 2.2E+05 UGT76G1var56
L246F_A352G_S357N_Q432E 0.4 4.1E+04 UGT76G1var57
S241I_T263S_L379G_A385T 0.8 1.5E+05 UGT76G1var58
S357C_S375M_N409R_Q425E 7.5 2.2E+06 UGT76G1var59
I335V_K393G_V394I_Y397Q 33.0 2.7E+07 UGT76G1var60
E231A_L243A_V279I_S357N 0.5 9.5E+04 UGT76G1var61
I43V_F265I_Q266S_L379A 6.4 2.0E+06 UGT76G1var62
L243P_P272A_V394I_V396F 0.1 3.4E+04 UGT76G1var63
F314S_R334H_Q342K_L379G 3.4 1.2E+06 UGT76G1var64
F22L_A239I_R334H_I407V 0.3 3.1E+04 UGT76G1var65
A33G_A239V_P382M_Q425E 1.2 3.3E+05 UGT76G1var66
F265L_V310I_V366I_A385T 0.8 3.7E+05 UGT76G1var67
E224D_F314S_S375R_Y397E -2.1 -5.6E+05 UGT76G1var68
Q342K_G348P_I373L_Y397E -1.4 -1.1E+05 UGT76G1var69
S274G_I295T_I335V_L379A 24.7 8.3E+06 UGT76G1var70
E224A_I295T_V300A_G348P 24.0 8.4E+06 UGT76G1var71
I295M_V300A_K393G_Q432E 42.9 2.1E+07 UGT76G1var72
T284L_D301N_K303G_S375R 19.2 9.1E+06 UGT76G1var73
F22Y_D301N_R334H_Q342E_V396F 0.8 8.7E+05 UGT76G1var74
I295T_I373L_S375R_Y397Q_Q432E 0.6 9.6E+04 UGT76G1var75
F41L_A239I_Q266S_S375M_P382M 0.8 -1.3E+05 UGT76G1var76
F22Y_A239I_L246F_I295M_R334K 2.6 7.2E+05 UGT76G1var77
A239V_F265I_I295T_D301N_K393G 1.9 4.4E+05 UGT76G1var78
V279I_V300A_V310I_I335V_S357C 3.2 8.2E+05 UGT76G1var79
E224D_T284I_V366I_I373L_K393G 8.5 3.8E+06 UGT76G1var80
L243P_L379A_S389E_Q425E_Q432E 1.0 2.1E+05 UGT76G1var81
A33G_T263S_S274G_V279I_Y397E 15.0 6.5E+06 UGT76G1var82
E224D_L243A_F265L_R334H_A352G 1.1 2.5E+05 UGT76G1var83
I43V_Q342E_S357N_S375R_L379G 0.5 4.3E+04 UGT76G1var84
F22L_Q266S_F314S_A352G_S357C 1.2 2.3E+05 UGT76G1var85
T284L_G348P_F377C_P382M_N409R 1.8 4.0E+05 UGT76G1var86
E224A_T284L_V396F_Y397E_I407V 1.6 3.8E+05 UGT76G1var87
S241I_L243A_V300A_F314S_N409R 35.7 2.1E+07 UGT76G1var88
A239V_T284I_V310I_Q342K_L379A 1.6 3.8E+05 UGT76G1var89
F41L_E229A_E231A_F265L_P272A 1.2 2.1E+05 UGT76G1var90
E231A_S241I_S274G_Y397Q_Q425E 34.5 1.9E+07 UGT76G1var91
E224A_L246F_T263S_F265I_Q342K 1.2 2.3E+05 UGT76G1var92
K303G_S357N_V366I_V394I_I407V 1.6 3.6E+05 UGT76G1var93
I43V_Q266E_S375M_S389E_V394I 1.8 4.5E+05 UGT76G1var94
Q266E_P272A_R334K_G348P_L379G 72.0 7.9E+07 UGT76G1var95
A33G_I295M_K303G_I335V_A385T -1.3 -1.7E+05 UGT76G1var96
F22L_E229A_L243P_F377C_A385T 1.2 2.7E+05 *Mutations are noted as
follows: original amino acid-position-new amino acid: For example
the mutation of an alanine at position 33 to a glycine is noted as
A33G.
Example 27
[0454] In-vivo production of UGTSL2
[0455] UGTSL2 (GI_460410132/XP_004250485.1) amino acid
sequence:
TABLE-US-00035 MATNLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTRINLESIIKK
IPEKYADSIHLIELQLPELPELPPHYHTTNGLPPHLNPTLHKALKMSKPN
FSRILQNLKPDLLIYDVLQPWAEHVANEQNIPAGKLLTSCAAVFSYFFSF
RKNPGVEFPFPAIHLPEVEKVKIREILAKEPEEGGRLDEGNKQMMLMCTS
RTIEAKYIDYCTELCNWKVVPVGPPFQDLITNDADNKELIDWLGTKHENS
TVFVSFGSEYFLSKEDMEEVAFALELSNVNFIWVARFPKGEERNLEDALP
KGFLERIGERGRVLDKFAPQPRILNHPSTGGFISHCGWNSAMESIDFGVP
IIAMPIHNDQPINAKLMVELGVAVEIVRDDDGKIHRGEIAETLKSVVTGE
TGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK
[0456] The pET30A+ vector containing the UGTSL2 gene was introduced
in E. coli B121(DE3) by heat shock. The obtained cells were grown
in petri-dishes in the presence of Kanamycin and suitable colonies
were selected and allowed to grow in liquid LB medium (erlenmeyer
flasks). Glycerol was added to the suspension as cryoprotecteur and
400 .mu.l aliquots were stored at -20.degree. C. and at -80.degree.
C.
[0457] The storage aliquots of E. coli BL21(DE3) containing the
pET30A+_UGTSL2 plasmids were thawed and added to 30 mL of LBGKP
medium (20 g/L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50
mM Phosphate buffer pH 7.00; 2.5 g/L glucose and 50 mg/L of
Kanamycin). This culture was allowed to shake at 135 rpm at
30.degree. C. for 8 h.
[0458] The production medium contained 60 g/L of overnight express
instant TB medium (Novagen), 10 g/L of glycerol and 50 mg/L of
Kanamycin. The preculture was added to 200 mL of this medium and
the solution was allowed to stir at 20.degree. C. while taking
samples to measure the OD and pH. The culture gave significant
growth and a good OD was obtained. After 40 h, the cells were
harvested by centrifugation and frozen to obtain 6.22 g of cell wet
weight.
[0459] Lysis was performed on 1.4 g of cells by addition of
Bugbuster Master mix (Novagen) and the lysate was recovered by
centrifugation and used fresh.
Example 28
[0460] Determination of activity for Stevioside to Rebaudioside E
conversion with UGTSL and UGTSL2
[0461] UGTSL was prepared according to EXAMPLE 22, and UGTSL2 was
prepared according to EXAMPLE 27.
[0462] Activity tests were performed at 3 mL scale with 600 .mu.L
of lysate for the transformation of Stevioside using 0.5 mM of
substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl.sub.2 in 50 mM
Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed
by HPLC. HPLC Analysis. The HPLC assay was performed as described
in EXAMPLE 20.
[0463] The results for the different enzymes and the corresponding
chromatograms are provided below and shown in FIGS. 59a-b
TABLE-US-00036 Enzyme internal Stevioside conv..sup.1 Rebaudioside
E reference GI Number Version (reaction time) formation.sup.1 UGTSL
460409128 XP_004249992.1 74% (22 h.) 46% UGTSL2 460410132
XP_004250485.1 77% (2 h.) 50% Note: .sup.1Based on initial
concentration of Stevioside
Example 29
[0464] Determination of activity for Rubusoside to Rebaudioside E
conversion with UGTSL and UGTSL2
[0465] UGTSL was prepared according to EXAMPLE 22, and UGTSL2 was
prepared according to EXAMPLE 27.
[0466] Activity tests were performed at 3 mL scale with 600 .mu.L
of lysate for the transformation of Rubusoside using 0.5 mM of
substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl.sub.2 in 50 mM
Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed
by HPLC. The HPLC assay was performed as described in EXAMPLE
20.
[0467] The results for the different enzymes and the corresponding
chromatograms are provided below and shown in FIGS. 60a-b.
TABLE-US-00037 Enzyme internal Rubusoside conv..sup.1 Rebaudioside
E reference GI Number Version (reactiontime) formation.sup.1 UGTSL
460409128 XP_004249992.1 70% (45 h.) 27% UGTSL2 460410132
XP_004250485.1 80% (2 h.) 55% Note: .sup.1Based on initial
concentration of Rubusoside
Example 30
[0468] Determination of activity for Rebaudioside A to Rebaudioside
D conversion with UGTSL2
[0469] UGTSL2 was prepared according to EXAMPLE 27.
[0470] Activity tests were performed at 3 mL scale with 60 .mu.L of
lysate for the transformation of Rebaudioside A using 0.5 mM of
substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl.sub.2 in 50 mM
Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed
by HPLC. The HPLC assay was performed as described in EXAMPLE
20.
[0471] The result after 23 h. of reaction and the corresponding
chromatogram is provided below and shown in FIG. 61.
TABLE-US-00038 Enzyme internal Rebaudioside A conv..sup.1
Rebaudioside D reference GI Number Version (reaction time)
formation.sup.1 UGTSL2 460410132 XP_004250485.1 78% (23 h.) 75%
Note: .sup.1Based on initial concentration of Rebaudioside A
Example 31
[0472] Identification of Glycosides
[0473] The reaction mixtures prepared according to EXAMPLE 30 and
incubated for 45 hrs was analyzed by LC-MS, along with Stevia
rebaudiana Bertoni leaf extract "MLD1" produced by PureCircle Sdn
Bhd (Malaysia), to determine the occurrence of formed glycosides in
nature.
[0474] An Agilent 1200 series HPLC system, equipped with binary
pump (G1312B), autosampler (G1367D), thermostatted column
compartment (G1316B), DAD detector (G1315C), connected with Agilent
6110A MSD, and interfaced with "LC/MSD Chem station" software, was
used.
[0475] Instrument Conditions
TABLE-US-00039 Column Phenomenex Prodigy 3u C18 100 A, 4.6 mm
.times. 250 mm, 3 .mu.m Column Temperature 55.degree. C. Detection
DAD at 210 nm bw 360 nm MSD (Scan and SIM mode) Mode: ES-API,
Negative Polarity Drying gas flow: 13.0 L/min Nebulizer pressure:
30 psig Drying gas temperature: 270.degree. C. Analysis duration 75
min Injected volume 10 .mu.L Flow rate 0.5 mL/min
[0476] Mobile Phase Gradient Program
TABLE-US-00040 Time (min) A (%): Formic acid 0.1% B (%):
Acetonitrile 0 75 25 30 75 25 33 68 32 75 68 32
[0477] The assay shown in FIG. 62 shows that the compound observed
on LC-MS system at 11.77 min is the same as the compound at 3.5
min, in EXAMPLE 23 (C.sub.56H.sub.90O.sub.33; later confirmed as
reb M2), and the compound observed at 26.64 min is the same as the
compound at 7.6 min, in EXAMPLE 23 (C.sub.50H.sub.80O.sub.28; reb
UNK; later confirmed as reb D2). Other isomers of reb X were
observed at 13.96 min and also another isomer form of reb D was
observed at 25.06 min. All observed compounds occurred in the
extract of Stevia rebaudiana Bertoni plant.
Example 32
[0478] In vivo preparation and activity determination of UGTLB
[0479] UGTLB (GI_209954733/BAG80557.1) amino acid sequence
TABLE-US-00041 mGTEVTVHKNTLRVLMFPWLAYGHISPFLNVAKKLVDRGFLIYLCSTAI
NLKSTIKKIPEKYSDSIQLIELHLPELPELPPHYHTTNGLPPHLNHTLQ
KALKMSKPNFSKILQNLKPDLVIYDLLQQWAEGVANEQNIPAVKLLTSG
AAVLSYFFNLVKKPGVEFPFPAIYLRKNELEKMSELLAQSAKDKEPDGV
DPFADGNMQVMLMSTSRIIEAKYIDYFSGLSNWKVVPVGPPVQDPIADD
ADEMELIDWLGKKDENSTVFVSFGSEYFLSKEDREEIAFGLELSNVNFI
WVARFPKGEEQNLEDALPKGFLERIGDRGRVLDKFAPQPRILNHPSTGG
FISHCGWNSVMESVDFGVPIIAMPIHLDQPMNARLIVELGVAVEIVRDD
YGKIHREEIAEILKDVIAGKSGENLKAKMRDISKNLKSIRDEEMDTAAE ELIQLCKNSPKLK
[0480] The pET30A+ vector containing the UGTLB gene was introduced
in E. coli B121(DE3) by heat shock. The obtained cells were grown
in petri-dishes in the presence of Kanamycin and suitable colonies
were selected and allowed to grow in liquid LB medium (erlenmeyer
flasks). Glycerol was added to the suspension as cryoprotecteur and
400 .mu.L aliquots were stored at -20.degree. C. and at -80.degree.
C.
[0481] The storage aliquots of E. coli BL21(DE3) containing the
pET30A+_UGTLB plasmids were thawed and added to 30 mL of LBGKP
medium (20 g/L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50
mM Phosphate buffer pH 7.00; 2.5 g/L glucose and 50 mg/L of
Kanamycine). This culture was allowed to shake at 135 rpm at
30.degree. C. for 8 h.
[0482] The production medium contained 60 g/L of overnight express
instant TB medium (Novagen), 10 g/L of glycerol and 50 mg/L of
Kanamycine. The preculture was added to 200 mL of this medium and
the solution was allowed to stir at 20.degree. C. while taking
samples to measure the OD and pH. The culture gave significant
growth and a good OD was obtained. After 40 h, the cells were
harvested by centrifugation and frozen to obtain 5.7 g of cell wet
weight.
[0483] Lysis was performed on 1.2 g of cells by addition of 6 mL
Bugbuster Master mix (Novagen) and the lysate was recovered by
centrifugation and used fresh.
[0484] Determination of Activity for Stevioside to Rebaudioside E
Conversion with UGTLB
[0485] Activity tests were performed at 3 mL scale with 600 .mu.L
of lysate for the transformation of Stevioside using 0.5 mM of
substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl.sub.2 in 50 mM
Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed
by HPLC. The corresponding chromatograms are depicted in FIG.
63a.
TABLE-US-00042 Enzyme Stevioside internal conv..sup.1 Rebaudioside
E reference GI Number Version (reaction time) formation.sup.1 UGTLB
209954733 BAG80557.1 89% (22 h.) 3% Note: .sup.1Based on initial
concentration of Stevioside
[0486] Determination of Activity for Rubusoside to Rebaudioside E
Conversion with UGTLB
[0487] Activity tests were performed at 3 mL scale with 600 .mu.L
of lysate for the transformation of Rubusoside using 0.5 mM of
substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl.sub.2 in 50 mM
Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed
by HPLC. The corresponding chromatograms are depicted in FIG.
63b.
TABLE-US-00043 Enzyme Rubusoside internal conv..sup.1 Rebaudioside
E reference GI Number Version (reaction time) formation.sup.1 UGTLB
209954733 BAG80557.1 65% (5 h.) 4% Note: .sup.1Based on initial
concentration of Rubusoside
[0488] Determination of Activity for Rebaudioside A to Rebaudioside
D Conversion with UGTLB
[0489] Activity tests were performed at 3 mL scale with 600 .mu.L
of lysate for the transformation of Rebaudioside A using 0.5 mM of
substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl.sub.2 in 50 mM
Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed
by HPLC. The corresponding chromatogram after 23 h. of reaction is
depicted in FIG. 63c.
TABLE-US-00044 Enzyme Rebaudioside Rebaudioside internal A
conv..sup.1 D reference GI Number Version (reaction time)
formation.sup.1 UGTLB 209954733 BAG80557.1 72% (22 h.) 10% Note:
.sup.1Based on initial concentration of Rebaudioside A
Example 33
[0490] Determination of reaction products for Rubusoside and
Stevioside conversion with UGTSL, UGTSL2, and UGTLB
[0491] Conversion of stevioside with UGTSL and UGTSL2 was conducted
in similar manner to Example 28, and the conversion of rubusoside
with UGTSL and UGTSL2 was conducted similarly to Example 29.
Conversions of rubusoside and stevioside with UGTLB was conducted
similarly to Example 32.
[0492] The reaction mixtures were analyzed by LCMS to determine all
reaction products.
Rubusoside Conversion Products
TABLE-US-00045 [0493] LC-MS, peak area ratio (%) Unknown peak
Unknown peak Unknown peak Sample UGT (reaction #1 (MW804) #2
(MW804) #3 (MW804) ID time) Rub Stev REb E Reb D RT 30.70 min RT
49.50 min RT 50.40 min S151N15 UGTSL2 (2 hrs) 3.54 2.12 52.88 6.73
12.02 9.94 12.77 S151N17 UGTLB (5 hrs) 13.49 ND 9.21 1.29 4.07
66.67 5.27 S151N22 UGTSL (45 hrs) 7.82 2.37 35.88 3.45 20.38 27.75
2.35
Stevioside Conversion Products
TABLE-US-00046 [0494] LC-MS, peak area ratio (%) Unknown peak
Unknown peak Unknown peak UGT (reaction #1 (MW966) #2 (MW966) #3
(MW966) Sample ID time) Stev Reb E Reb D RT = 22.60 min RT = 26.50
min RT = 29.50 min S151N26 UGTSL2 (2 hrs) 20.01 42.56 1.70 4.48
5.56 25.70 S151N28 UGTLB (2 hrs) 43.11 3.12 ND ND 53.78 ND S151N33
UGTSL (22 hrs) 25.24 49.68 0.54 3.97 20.56 ND
[0495] It can be seen that amongst Rubusoside conversion products,
besides Stevioside, Reb E and Reb D, there are at least 3
additional compounds with Molecular Weight of 804. The retention
time of these compounds do not match with Reb B which is known to
have same Molecular Weight as Stevioside. Since these compounds
have same molecular weight with Stevioside it can be assumed that
these novel steviol glycosides are isomers of Stevioside. On the
other hand amongst Stevioside conversion products, besides Reb E
and Reb D, there are at least 3 additional compounds with Molecular
Weight of 966. The retention time of these compounds do not match
with Reb A which is known to have same Molecular Weight as Reb E.
Since these compounds have same molecular weight with Reb A and Reb
E it can be assumed that these novel steviol glycosides are isomers
of Reb A (Reb E).
Example 34
[0496] In Vivo Production of UGT76G1 in S. cerevisiae
[0497] UGT76G1 [Stevia rebaudiana] (gi_37993653/gb_AAR06912.1)
TABLE-US-00047 MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNF
NKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADE
LRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLF
NFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQIL
KEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHL
TASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLV
DSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAI
GAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLEN
GWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLES LVSYISSL
[0498] The above mentioned amino acid sequence was codon optimized
for expression in S. cerevisiae. Furthermore the yeast consensus
sequence AACACA was added before the ATG start codon. The synthetic
gene was subcloned in the pYES2 vector using Hind III and Xba I
restriction sites. The pYES2_UGT76G1_.delta..sub.C vector was used
to transform chemically competent S. cerevisiae INVSc1 cells
(Invitrogen).
[0499] The cells were grown on a solid synthetic minimal medium
containing 2% glucose lacking Uracil and a single colony was picked
and allowed to grow in liquid synthetic minimal medium lacking
Uracil (SC-U containing 2% glucose). After centrifugation, the
cells were suspended with SC-U (containing 2% glucose) and 60%
glycerol/water. Aliquots were stored at -80.degree. C. and one
aliquot was used to start a culture in SC-U (containing 2% glucose)
for 43 h at 30.degree. C. Part of this culture was centrifuged and
suspended in induction medium (SC-U containing 2% galactose) for
19h30 at 30.degree. C.
[0500] Cells were obtained by centrifugation and lysis with five
volumes of CelLytic.TM. Y Cell Lysis Reagent (Sigma). The lysates
were used directly for activity testing (UGT76G1_Sc).
Example 35
[0501] Determination of activity of UGT76G1_.delta..sub.C for the
conversion of Rebaudioside D to Rebaudioside M
[0502] UGT76G1_.delta..sub.C was prepared according to EXAMPLE 34.
Activity tests were performed at 2 mL scale with 200 .mu.L of
lysate for the transformation of Rebaudioside D using 0.5 mM of
substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl.sub.2 in 50 mM
Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed
by HPLC. The corresponding chromatogram is depicted in FIG. 64.
TABLE-US-00048 Enzyme Rebaudioside D internal reference conv..sup.1
(reaction time) Rebaudioside M selectivity.sup.1 UGT76G1_Sc 85% (21
h.) 100% Note: .sup.1Based on initial concentration of Rebaudioside
D
Example 36
[0503] In vivo production of UGTSL in S. cerevisiae
[0504] UGTSL [Solanum lycopersicum]
(gi_460409128/XP_004249992.1
TABLE-US-00049 MSPKLHKELFFHSLYKKTRSNHTMATLKVLMFPFLAYGHISPYLNVAKKL
ADRGFLIYFCSTPINLKSTIEKIPEKYADSIHLIELHLPELPQLPPHYHT
TNGLPPNLNQVLQKALKMSKPNFSKILQNLKPDLVIYDILQRWAKHVANE
QNIPAVKLLTSGAAVFSYFFNVLKKPGVEFPFPGIYLRKIEQVRLSEMMS
KSDKEKELEDDDDDDDLLVDGNMQIMLMSTSRTIEAKYIDFCTALTNWKV
VPVGPPVQDLITNDVDDMELIDWLGTKDENSTVFVSFGSEYFLSKEDMEE
VAFALELSNVNFIWVARFPKGEERNLEDALPKGFLERIGERGRVLDKFAP
QPRILNHPSTGGFISHCGWNSAMESIDFGVPIIAMPMHLDQPMNARLIVE
LGVAVEIVRDDDGKIHRGEIAETLKGVITGKTGEKLRAKVRDISKNLKTI
RDEEMDAAAEELIQLCRNGN
[0505] The above mentioned amino acid sequence was codon optimized
for expression in S. cerevisiae. Furthermore the yeast consensus
sequence AACACA was added before the ATG start codon. The synthetic
gene was subcloned in the pYES2 vector using Hind III and Xba I
restriction sites. The pYES2_UGTSL_.delta..sub.C vector was used to
transform chemically competent S. cerevisiae INVSc1 cells
(Invitrogen).
[0506] The cells were grown on a solid synthetic minimal medium
containing 2% glucose, lacking Uracil and a single colony was
picked and allowed to grow in liquid synthetic minimal medium
lacking Uracil (SC-U containing 2% glucose). After centrifugation,
the cells were suspended with SC-U (containing 2% glucose) and 60%
glycerol/water. Aliquots were stored at -80.degree. C. and one
aliquot was used to start a culture in SC-U (containing 2% glucose)
for 43 h at 30.degree. C. Part of this culture was centrifuged and
suspended in induction medium (SC-U containing 2% galactose) for
19h30 at 30.degree. C. Cells were obtained by centrifugation and
lysis with five volumes of CelLytic.TM. Y Cell Lysis Reagent
(Sigma). The lysates were used directly for activity testing
(UGTSL_Sc).
Example 37
[0507] Determination of activity of UGTSL_.delta..sub.C for the
conversion of Rebaudioside A to Rebaudioside D
[0508] UGTSL_.delta..sub.C was prepared according to EXAMPLE 36.
Activity tests were performed at 2 mL scale with 200 .mu.L of
lysate for the transformation of Rebaudioside A using 0.5 mM of
substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl.sub.2 in 50 mM
Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed
by HPLC. The corresponding chromatogram is depicted in FIG. 65.
TABLE-US-00050 Enzyme Rebaudioside A internal reference conv..sup.1
(reaction time) Rebaudioside D selectivity.sup.1 UGTSL_Sc 46% (4 h)
42% Note: .sup.1Based on initial concentration of Rebaudioside
A
Example 38
[0509] Isolation of Rebaudioside M
[0510] The amount of the product mixture of Example 14 was not
large enough to separate via preparative HPLC methods. Accordingly,
analytical HPLC with a series of injections was used to separate
the components of the mixture. Separation was conducted according
to the method described above in Example 14 to provide two
fractions corresponding to the two main peaks in the HPLC trace of
FIG. 5: Fraction A (retention time 24.165 minutes) and Fraction B
(retention time 31.325 minutes).
[0511] The retention time of Fraction A was consistent with reb D,
indicating unreacted starting material from the biotransformation
reaction.
[0512] The retention time of purified Fraction B (FIG. 6) was
consistent with reb M, indicating successful biotransformation from
reb D. The identity of the material collected in Fraction B as reb
M was confirmed by co-injection of purified Fraction B with a reb M
standard (available from PureCircle, HPLC trace of reb M standard
shown in FIG. 7). Both Fraction B and the reb M standard were found
to elute at the same retention time (FIG. 8), indicating Fraction B
was reb M
[0513] The identity of Fraction B as reb M was also separately
confirmed by NMR and HRMS. For sampling, Fraction B was
concentrated under rotary evaporator, freeze dried and dried for 40
h at 40.degree. C.
[0514] The NMR sample was dissolved in deuterated pyridine
(C.sub.5D.sub.5N) and spectra were acquired on a Varian Unity Plus
600 MHz instrument using standard pulse sequences. The NMR spectra
of Fraction B was compared to the NMR spectra of reb M An overlay
of the two spectra (FIG. 9) showed consistency of peaks of Fraction
B with reb M A table of the NMR assignments for reb M is shown
below:
TABLE-US-00051 .sup.1H and .sup.13C NMR spectral data for
Rebaudioside M in C.sub.5D.sub.5N.sup.a-c. Position .sup.13C NMR
.sup.1H NMR 1 40.3 0.75 t (13.2) 1.76 m 2 19.6 1.35 m 2.24 m 3 38.4
1.01 m 2.30 d (13.3) 4 44.3 -- 5 57.4 1.06 d (12.8) 6 23.5 2.23 m
2.41 q (13.2) 7 42.6 1.41 m 1.80 m 8 41.2 -- 9 54.3 0.91 d (7.7) 10
39.7 -- 11 20.2 1.65 m 1.75 m 12 38.5 1.86 m 2.73 m 13 87.6 -- 14
43.3 2.02 m 2.74 m 15 46.5 1.88 d (16.4) 2.03 m 16 153.3 -- 17
104.9 4.90 s 5.69 s 18 28.2 1.32 s 19 176.9 -- 20 16.8 1.38 s 1'
94.9 6.39 d (8.2) 2' 76.9 4.51 t (8.5) 3' 88.6 5.09 t (8.5) 4' 70.1
4.18 m 5' 78.4 4.13 m 6' 61.8 4.20 m 4.31 m 1'' 96.2 5.46 d (7.1)
2'' 81.4 4.13 m 3'' 87.9 4.98 t (8.5) 4'' 70.4 4.07 t (9.6) 5''
77.7 3.94 m 6'' 62.6 4.19 m 4.32 m 1''' 104.8 5.48 d (7.7) 2'''
75.8 4.15 m 3''' 78.6 4.13 m 4''' 73.2 3.98 m 5''' 77.6 3.74 ddd
(2.8, 6.4, 9.9) 6''' 64.0 4.27 m 4.51m 1'''' 103.9 5.45 d (7.5)
2'''' 75.6 3.98 m 3'''' 77.8 4.50 t (7.8) 4'''' 71.3 4.14 m 5''''
78.0 3.99 m 6'''' 62.1 4.20 m 4.32 m 1''''' 104.2 5.81 d (7.2)
2''''' 75.5 4.20 m 3''''' 78.4 4.20 m 4''''' 73.6 4.10 m 5'''''
77.8 3.90 ddd (2.8, 6.4, 9.9) 6''''' 64.0 4.32 m 4.64 d (10.3)
1'''''' 104.1 5.31 d (8.0) 2'''''' 75.5 3.95 m 3'''''' 78.0 4.37 t
(9.1) 4'''''' 71.1 4.10 m 5'''''' 78.1 3.85 ddd (1.7, 6.1, 9.9)
6'''''' 62.1 4.10 m 4.32 m .sup.aassignments made on the basis of
COSY, HMQC and HMBC correlations; .sup.bChemical shift values are
in .delta. (ppm); .sup.cCoupling constants are in Hz.
[0515] HRMS (FIG. 10) was generated with a Waters Premier
Quadropole Time-of-Flight (Q-TOF) mass spectrometer equipped with
an electrospray ionization source operated in the positive-ion
mode. The sample was dissolved in methanol and eluted in 2:2:1
methanol: acetonitrile: water and introduced via infusion using the
onboard syringe pump. The presence of reb M was confirmed by a
[M+Na].sup.+ adduct at m/z 1313.5265, which corresponds to a
molecular formula of C.sub.56H.sub.90O.sub.33
##STR00008##
Example 39
[0516] Isolation and Characterization of Reb D2
[0517] Crude Reaction Sample.
[0518] The sample, Lot CB-2977-106, used for isolation, was
prepared according to Example 22 with UGTSL (GI #460409128).
[0519] HPLC Analysis.
[0520] Preliminary HPLC analyses of samples were performed using a
Waters 2695 Alliance System with the following method: Phenomenex
Synergi Hydro-RP, 4.6.times.250 mm, 4 .mu.m (p/n 00G-4375-E0);
Column Temp: 55.degree. C.; Mobile Phase A: 0.0284% ammonium
acetate (NH.sub.4OAc) and 0.0116% acetic acid (HOAc) in water;
Mobile Phase B: Acetonitrile (MeCN); Flow Rate: 1.0 mL/min;
Injection volume: 10 .mu.L. Detection was by UV (210 nm) and
CAD.
[0521] Gradient:
TABLE-US-00052 Time (min) % A % B 0.0-8.5 75 25 10.0 71 29 16.5 70
30 18.5-24.5 66 34 26.5-29.0 48 52 31-37 30 70 38 75 25
[0522] Analyses of semi-preparative purification fractions were
performed with the following method: Waters Atlantis dC18,
4.6.times.100 mm, 5 .mu.m (p/n 186001340); Mobile Phase A: 25% MeCN
in water; Mobile Phase B: 30% MeCN in water; Flow Rate: 1.0 mL/min;
Injection volume: 10 .mu.L. Detection was by CAD.
[0523] Gradient:
TABLE-US-00053 Time (min) % A % B 0.0-5.0 100 0 20 20 80 25 20 80
30 100 0
[0524] LC-MS.
[0525] Preliminary analysis of the semi-synthetic steviol glycoside
mixture was carried out on a Waters AutoPurification HPLC/MS System
with a Waters 3100 Mass Detector operating in negative ion mode.
Analysis of the sample was performed using the following method:
Phenomenex Synergi Hydro-RP, 4.6.times.250 mm, 4 .mu.m (p/n
00G-4375-E0); Column Temp: 55.degree. C.; Mobile Phase A: 0.0284%
NH.sub.4OAc and 0.0116% HOAc in water; Mobile Phase B:
Acetonitrile; Flow Rate: 1.0 mL/min; Injection volume: 10 .mu.L.
Detection was by UV (210 nm), and MSD (-ESI m/z 500-2000). Gradient
conditions were as listed above.
[0526] Isolation by HPLC.
[0527] The purification was performed in two steps. The first
method used for the semi-preparative purification is summarized
below. Column: Waters Atlantis dC18, 30.times.100 mm, 5 .mu.m (p/n
186001375); Mobile Phase A: 25% MeCN in water; Mobile Phase B: 30%
MeCN in water; Flow Rate: 45 mL/min; Injection load: 160 mg
dissolved in 20 mL of water. Detection was by UV (205 nm).
[0528] Gradient:
TABLE-US-00054 Time (min) % A % B 0.0-5.0 100 0 20 20 80 25 20 80
30 100 0
[0529] The secondary purification used the same column and
conditions, but isocratic mobile phase: 20% MeCN in water.
[0530] Purification from Natural Extracts.
[0531] The purification was performed in three steps. The first
method used for the preparative purification is summarized below.
Primary Process: Waters Symmetry C18, 50.times.250 mm, 7 .mu.m (p/n
WAT248000); Isocratic mobile phase: 50% methanol (MeOH) in water
with 0.05% HOAc; Flow Rate: 85 mL/min; Injection load: 6 g crude
extract dissolved in 50 mL of mobile phase. Detection was by UV
(210 nm). Following the elution of target analytes, the column was
flushed with 85% MeOH in water.
[0532] Secondary Process: Waters Symmetry Shield RP18, 50.times.250
mm, 7 .mu.m (p/n WAT248000); Isocratic mobile phase: 20% MeCN in
water; Flow Rate: 100 mL/min; Injection load: 0.5 g primary
fraction dissolved in 30 mL of water. Detection was by UV (210
nm).
[0533] Tertiary Process: Waters Symmetry Shield RP18, 50.times.250
mm, 7 .mu.m (p/n WAT248000); Isocratic mobile phase: 20% MeCN in
water; Flow Rate: 100 mL/min; Injection load: 0.5 g secondary
fraction dissolved in 30 mL of water. Detection was by UV (210
nm).
[0534] MS and MS/MS.
[0535] MS and MS/MS data were generated with a Waters QT of Premier
mass spectrometer equipped with an electrospray ionization source.
Samples were analyzed by negative ESI. Samples were diluted with
H.sub.2O:acetonitrile (1:1) by 50 fold and introduced via infusion
using the onboard syringe pump. The samples were diluted to yield
good s/n which occurred at an approximate concentration of 0.01
mg/mL.
[0536] NMR.
[0537] The sample was prepared by dissolving 1-2 mg in 150 .mu.L of
pyridine-d.sub.5 and NMR data were acquired on a Bruker Avance 500
MHz instrument with a 2.5 mm inverse detection probe. The .sup.1H
NMR spectrum was referenced to the residual solvent signal
(.delta..sub.H 8.74 and .delta..sub.C 150.35 for
pyridine-d.sub.5).
[0538] Results and Discussion
[0539] Isolation and Purification.
[0540] Isolation was performed on steviol glycoside mixture, Lot
number CB-2977-106, prepared according to Example 22 with UGTSL (GI
#460409128) The material was analyzed by LC-MS using the method
described above and results are provided in FIG. 11. The targeted
peak of interest was that at 7.7 min in the TIC chromatogram. The
mass spectrum of this peak provided a [M-H].sup.- ion at m/z
1127.6. The provided sample was preliminarily processed in a single
injection (160 mg) using the first method condition provided above.
This method fractionated the material into `polar` and `non-polar`
mixtures of glycosides. The `polar` mixture was then reprocessed
using the second-step conditions above. The semi-preparative HPLC
trace is provided in FIG. 12. From this semi-preparative
collection, the compound was isolated with a purity >99% (CAD,
AUC). The fraction analysis is provided in FIG. 13. Following the
purification, the combined fractions were concentrated by rotary
evaporation at 35.degree. C. and lyophilized. Approximately 1-2 mg
was obtained for characterization.
[0541] Mass Spectrometry.
[0542] The ESI-TOF mass spectrum acquired by infusing a sample
showed a [M-H].sup.- ion at m/z 1127.4709. The mass of the
[M-H].sup.- ion was in good agreement with the molecular formula
C.sub.50H.sub.80O.sub.28 (calcd for C.sub.50H.sub.79O.sub.28:
1127.4758, error: -4.3 ppm). The MS data confirmed a nominal mass
of 1128 Daltons with the molecular formula,
C.sub.50H.sub.80O.sub.28.
[0543] The MS/MS spectrum (selecting the [M-H].sup.- ion at m/z
1127.5 for fragmentation) indicated the loss of two glucose units
and sequential loss of three glucose moieties at m/z 641.3187,
479.2655 and 317.2065.
[0544] NMR Spectroscopy.
[0545] A series of NMR experiments including .sup.1H NMR (FIG. 14),
.sup.13C NMR (FIGS. 15 and 16), .sup.1H-.sup.1H COSY (FIG. 17),
HSQC-DEPT (FIG. 18), HMBC (FIGS. 19 and 20), and 1D-TOCSY were
performed to allow assignment of the compound.
[0546] The .sup.1H, .sup.1H-.sup.1H COSY, .sup.1H-.sup.13C
HSQC-DEPT and .sup.1H-.sup.13C HMBC NMR data indicated that the
central core of the glycoside is a diterpene. The presence of five
anomeric protons observed in the .sup.1H and .sup.1H-.sup.13C
HSQC-DEPT spectra confirm five sugar units in the structure. The
methylene .sup.13C resonance at .delta..sub.C 69.9 in the
.sup.1H-.sup.13C HSQC-DEPT spectrum indicated the presence of a
1.fwdarw.6 sugar linkage in the structure. The linkages of sugar
units were assigned using .sup.1H-.sup.13C HMBC and 1D-TOCSY
correlations.
[0547] A HMBC correlation from the methyl protons at .delta..sub.H
1.29 to the carbonyl at .delta..sub.C 177.7 allowed assignment of
one of the tertiary methyl groups (C-18) as well as C-19 and
provided a starting point for the assignment of the rest of the
aglycone. Additional HMBC correlations from the methyl protons
(H-18) to carbons at .delta..sub.C 38.9, 45.0, and 57.8 allowed
assignment of C-3, C-4, and C-5. Analysis of the .sup.1H-.sup.13C
HSQC-DEPT data indicated that the carbon at .delta..sub.C 38.9 was
a methylene group and the carbon at .delta..sub.C 57.8 was a
methine which were assigned as C-3 and C-5, respectively. This left
the carbon at .delta..sub.C 45.0, which did not show a correlation
in the HSQC-DEPT spectrum, to be assigned as the quaternary carbon,
C-4. The .sup.1H chemical shifts for C-3 (.delta..sub.H 0.98 and
2j.36) and C-5 (.delta..sub.H 1.04) were assigned using the
HSQC-DEPT data. A COSY correlation between one of the H-3 protons
(.delta..sub.H 0.98) and a proton at .delta..sub.H 1.43 allowed
assignment of one of the H-2 protons which in turn showed a
correlation with a proton at .delta..sub.H 0.75 which was assigned
to C-1. The remaining .sup.1H and .sup.13C chemical shifts for C-1
and C-2 were then assigned on the basis of additional COSY and
HSQC-DEPT correlations and are summarized in the following
table.
TABLE-US-00055 .sup.1H and .sup.13C NMR (500 and 125 MHz,
pyridine-d.sub.5), Assignments of Reb D2. Reb D2 Position .sup.13C
.sup.1H 1 41.3 0.75 t (11.0) 1.76 m 2 19.9 1.43 m 2.20 m 3 38.9
0.98 m 2.36 d (12.1) 4 45.0 -- 5 57.8 1.04 d (12.5) 6 22.7 1.92 m
2.43 m 7 42.2 1.22 m 1.30 m 8 43.1 -- 9 54.5 0.88 brs 10 40.3 -- 11
21.1 1.65 m 1.69 m 12 37.5 1.99 m 2.25 m 13 87.1 -- 14 44.5 1.80 d
(11.7) 2.65 d (11.7) 15 48.3 1.31 m 2.04 brs 16 154.7 -- 17 105.2
5.01 s 5.64 s 18 28.8 1.29 s 19 177.7 -- 20 16.0 1.30 s
[0548] The other tertiary methyl singlet, observed at .delta..sub.H
1.30 showed HMBC correlations to C-1 and C-5 and was assigned as
C-20. The methyl protons showed additional HMBC correlations to a
quaternary carbon (.delta..sub.C 40.3) and a methine carbon
(.delta..sub.C 54.5) which were assigned as C-10 and C-9,
respectively. COSY correlations between H-5 (.delta..sub.H 1.04)
and protons at .delta..sub.H 1.92 and 2.43 then allowed assignment
of the H-6 protons which in turn showed correlations to protons at
.delta..sub.H 1.22 and 1.30 which were assigned to C-7. The
.sup.13C chemical shifts for C-6 (.delta..sub.C 22.7) and C-7
(.delta..sub.C 42.2) were then determined from the HSQC-DEPT data.
COSY correlations between H-9 (.delta..sub.H 0.88) and protons at
.delta..sub.H 1.65 and 1.69 allowed assignment of the H-11 protons
which in turn showed COSY correlations to protons at .delta..sub.H
1.99 and 2.25 which were assigned as the H-12 protons. The
HSQC-DEPT data was then used to assign C-11 (.delta..sub.C 21.1)
and C-12 (.delta..sub.C 37.5). HMBC correlations from the H-12
proton (.delta..sub.H 2.25) to carbons at .delta..sub.C 87.1 and
154.7 allowed assignment of C-13 and C-16, respectively. The
olefinic protons observed at .delta..sub.H 5.01 and 5.64 showed
HMBC correlations to C-13 and were assigned to C-17 (.delta..sub.C
105.2 via HSQC-DEPT). The olefinic protons H-17 and the methine
proton H-9 showed HMBC correlations to a carbon at .delta..sub.C
48.3 which was assigned as C-15. An additional HMBC correlation
from H-9 to a methylene carbon at .delta..sub.C 44.5 then allowed
assignment of C-14. The .sup.1H chemical shifts at C-14
(.delta..sub.H 1.80 and 2.65) and C-15 (.delta..sub.H 1.31 and
2.04) were assigned using the HSQC-DEPT data.
[0549] The key HMBC and COSY correlations used to assign the
aglycone region are provided below:
##STR00009##
[0550] Analysis of the .sup.1H-.sup.13C HSQC-DEPT data confirmed
the presence of five anomeric protons. Three of the anomeric
protons were well resolved at .delta..sub.H 6.02 (.delta..sub.C
96.1), 5.57 (Sc 105.3), and 5.34 (.delta..sub.C 105.3) in the
.sup.1H NMR spectrum. The remaining two anomeric protons observed
at .delta..sub.H 5.04 (.delta..sub.C 105.6) and 5.07 (.delta..sub.C
98.7) which were obscured by solvent (HOD) resonance in the .sup.1H
spectrum were identified by .sup.1H-.sup.13C HSQC-DEPT data. The
anomeric proton observed at .delta..sub.H 6.02 showed HMBC
correlation to C-19 which indicated that it corresponds to the
anomeric proton of Glc.sub.I. Similarly, the anomeric proton
observed at .delta..sub.H 5.07 showed an HMBC correlation to C-13
allowing it to be assigned as the anomeric proton of
Glc.sub.II.
[0551] The Glc.sub.I anomeric proton (.delta..sub.H 6.02) showed a
COSY correlation to a proton at .delta..sub.H 4.07 was assigned as
Glc.sub.I H-2 which in turn showed a COSY correlation to a proton
at .delta..sub.H 4.22 (Glc.sub.I H-3) which showed a COSY
correlation with a proton at .delta..sub.H 4.12 (Glc.sub.I H-4).
Due to data overlap, the COSY spectrum did not allow assignment of
H-5 or the H-6 protons. Therefore, a series of 1D-TOCSY experiments
were performed using selective irradiation of the Glc.sub.I
anomeric proton with several different mixing times. In addition to
confirming the assignments for Glc.sub.I H-2 through H-4, the
1D-TOCSY data showed a proton at .delta..sub.H 4.04 assigned as
Glc.sub.I H-5 and a proton at .delta..sub.H 4.68 assigned as one of
the Glc.sub.I H-6 protons. The latter proton was also used for
1D-TOCSY experiments. The selective irradiation of H-6 with several
different mixing times also confirmed the assignment of Glc.sub.I
H-1 to H-5 as well as the remaining methylene proton of H-6
(.delta..sub.H 4.30). Assignment of the .sup.13C chemical shifts
for Glc.sub.I C-2 (.delta..sub.C 74.2), C-3 (.delta..sub.C 79.1),
C-4 (.delta..sub.C 72.1), C-5 (Sc 78.5), and C-6 (.delta..sub.C
69.9) was determined using the .sup.1H-.sup.13C HSQC-DEPT data to
complete the assignment of Glc.sub.I. Furthermore, the presence of
a methylene .sup.13C resonance at .delta..sub.C 69.9 in the
.sup.1H-.sup.13C HSQC-DEPT spectrum indicated a 1.fwdarw.6 sugar
linkage of Glc.sub.I in the structure.
[0552] Out of four remaining unassigned glucose moieties, one was
assigned as a substituent at C-6 of Glc.sub.I on the basis of
.sup.1H-.sup.13C HSQC-DEPT, HMBC, and 1D-TOCSY correlations. The
relatively downfield shift of a methylene .sup.13C resonance of
Glc.sub.I at .delta..sub.C 69.9 in the HSQC-DEPT spectrum indicated
a 1.fwdarw.6 sugar linkage of Glc.sub.I. The anomeric proton
observed at .delta..sub.H 5.04 showed HMBC correlation to Glc.sub.I
C-6 and was assigned as the anomeric proton of Glc.sub.V.
Similarly, methylene protons of Glc.sub.I showed HMBC correlations
to anomeric carbon of Glc.sub.V confirming the presence of a
1.fwdarw.6 sugar linkage between Glc.sub.I and Glc.sub.V. The
Glc.sub.V anomeric proton showed a COSY correlation to a proton at
.delta..sub.H 4.00 which was assigned as Glc.sub.V H-2 which in
turn showed a COSY correlation to a proton at .delta..sub.H 4.22
(Glc.sub.V H-3). Due to data overlap, the COSY spectrum did not
allow assignment of Glc.sub.V H-4 based on the COSY correlation of
Glc.sub.V H-3. However, in the HMBC spectrum, Glc.sub.V H-3 showed
a correlation to Glc.sub.V C-5 (.delta..sub.C 78.9). In HSQC-DEPT
spectrum, Glc.sub.V C-5 showed a correlation to .delta..sub.H 3.89
(Glc.sub.V H-5). The Glc.sub.V H-5 showed COSY correlations to
.delta..sub.H 4.21, 4.37, and 4.48. In the HSQC-DEPT spectrum,
.delta..sub.H 4.21 showed a correlation to .delta..sub.C 71.4
(Glc.sub.V H-4), while .delta..sub.H 4.37 and 4.48 showed a
correlation to .delta..sub.C 63.1 and were assigned to Glc.sub.V
H-6a and H-6b, respectively. Assignment of the .sup.13C chemical
shifts for Glc.sub.V C-2 (.delta..sub.C 75.7) and C-3
(.delta..sub.C 79.1) was determined using the .sup.1H-.sup.13C
HSQC-DEPT data to complete the assignment of Glc.sub.V.
[0553] A summary of the .sup.1H and .sup.13C chemical shifts for
the glycoside at C-19 are shown in the following table:
TABLE-US-00056 .sup.1H and .sup.13C NMR (500 and 125 MHz,
pyridine-d.sub.5), Assignments of the reb D2 C-19 glycoside. Reb D2
Position .sup.13C .sup.1H Glc.sub.I-1 96.1 6.02 d (8.1) Glc.sub.I-2
74.2 4.07 m Glc.sub.I-3 79.1.sup.# 4.22 m.sup.# Glc.sub.I-4 72.1
4.12 m Glc.sub.I-5 78.5 4.04 m Glc.sub.I-6 69.9 4.30 m 4.68 d
(10.7) Glc.sub.V-1 105.6 5.04* Glc.sub.V-2 75.7 4.00 m Glc.sub.V-3
79.1.sup.# 4.22 m.sup.# Glc.sub.V-4 71.4 4.21 m Glc.sub.V-5 78.9
3.89 m Glc.sub.V-6 63.1 4.37 m 4.48 m *Anomeric proton was obscured
by solvent (HDO) resonance. Therefore, the coupling constant value
could not be determined. .sup.#1H and .sup.13C values can be
exchangeable between positions Glc.sub.I-3, Glc.sub.V-3 and
Glc.sub.IV-3.
[0554] A summary of the key HMBC, COSY, and 1D-TOCSY correlations
used to assign the C-19 glycoside region are provided below.
##STR00010##
[0555] Assignment of Glc.sub.II was carried out in a similar
manner. The Glc.sub.II anomeric proton (.delta..sub.H 5.07) showed
a COSY correlation to a proton at .delta..sub.H 4.37, assigned as
Glc.sub.II H-2, which in turn showed a COSY correlation to a proton
at .delta..sub.H 4.18 (Glc.sub.II H-3). This latter proton showed
an additional correlation with a proton at .delta..sub.H 3.88
(Glc.sub.II H-4) which also showed a COSY correlation to a proton
at .delta..sub.H 3.79 (Glc.sub.II H-5). Glc.sub.II H-5 also showed
a COSY correlation to Glc.sub.II H-6 protons (.delta..sub.H 4.08
and 4.46). Assignment of the .sup.13C chemical shifts for
Glc.sub.II C-2 (.delta..sub.C 81.3), C-3 (.delta..sub.C 88.4), C-4
(.delta..sub.C 71.1), C-5 (.delta..sub.C 77.9), and C-6
(.delta..sub.C 63.2) was determined using the HSQC-DEPT data. HMBC
correlations from Glc.sub.II H-3 to C-2 and C-4 and also from
Glc.sub.II H-4 to C-2 and C-5 confirmed the assignments made above.
Additional HMBC correlations of Glc.sub.II H-4 to Glc.sub.II C-6
further support to complete the assignment of Glc.sub.II.
[0556] Two of the remaining unassigned glucose moieties were
assigned as substituents at C-2 and C-3 of Glc.sub.II on the basis
of HMBC correlations. The anomeric proton observed at .delta..sub.H
5.57 showed a HMBC correlation to Glc.sub.II C-2 and was assigned
as the anomeric proton of Glc.sub.III. The anomeric proton observed
at .delta..sub.H 5.34 showed a HMBC correlation to Glc.sub.II C-3
and was assigned as the anomeric proton of Glc.sub.IV. The
reciprocal HMBC correlations from Glc.sub.II H-2 to the anomeric
carbon of Glc.sub.III and from Glc.sub.II H-3 to the anomeric
carbon of Glc.sub.IV were also observed.
[0557] The anomeric proton of Glc.sub.III (.delta..sub.H 5.57)
showed a COSY correlation with a proton at .delta..sub.H 4.19 which
was assigned as Glc.sub.III H-2. Due to data overlap, the COSY
spectrum did not allow assignment of H-3 to H-6 protons. Therefore,
a series of 1D-TOCSY experiments were performed using selective
irradiation of the Glc.sub.III anomeric proton with several
different mixing times. In addition to confirming the assignments
for Glc.sub.III H-2, the 1D-TOCSY data showed protons at
.delta..sub.H 4.24 (Glc.sub.III H-3), .delta..sub.H 4.27
(Glc.sub.III H-4), and .delta..sub.H 3.94 (Glc.sub.III H-5). Once
H-4 was assigned using 1D-TOCSY data, COSY correlations from H-4 to
H-5 and in turn to H-6 were used to assign H-6. In the COSY
spectrum, Glc.sub.III H-4 showed a correlation to Glc.sub.III H-5,
which in turn showed COSY correlations to .delta..sub.H 4.41 and
4.50 of Glc.sub.III H-6a and H-6b, respectively. The .sup.13C
chemical shifts for Glc.sub.III C-2 (.delta..sub.C 76.8), C-3
(.delta..sub.C 78.9), C-4 (.delta..sub.C 72.4), C-5 (.delta..sub.C
78.8), and C-6 (.delta..sub.C 63.5) were then determined using the
.sup.1H-.sup.13C HSQC-DEPT correlations to complete the assignment
of Glc.sub.III.
[0558] The anomeric proton of Glc.sub.IV (.delta..sub.H 5.34)
showed a COSY correlation with a proton at .delta..sub.H 4.06 which
was assigned as Glc.sub.IV H-2. Due to data overlap, the COSY
spectrum did not allow assignment of H-3 to H-6 protons. Therefore,
a series of 1D-TOCSY experiments were performed using selective
irradiation of the Glc.sub.IV anomeric proton with several
different mixing times. In addition to confirming the assignments
for Glc.sub.IV H-2, the 1D-TOCSY data showed protons at
.delta..sub.H 4.22 (Glc.sub.II, H-3), .delta..sub.H 4.18
(Glc.sub.IV H-4), and .delta..sub.H 4.10 (Glc.sub.II, H-5). Once
H-4 was assigned using 1D-TOCSY data, COSY correlations from H-4 to
H-5 and in turn to H-6 were used to assign H-6. In the COSY
spectrum, Glc.sub.II, H-4 showed a correlation to Glc.sub.IV, H-5,
which in turn showed COSY correlations to .delta..sub.H 4.32 and
4.58, Glc.sub.IV H-6a and H-6b, respectively. The .sup.13C chemical
shifts for Glc.sub.IV C-2 (.delta..sub.C 75.8), C-3 (.delta..sub.C
78.9), C-4 (.delta..sub.C 72.0), C-5 (.delta..sub.C 79.3), and C-6
(.delta..sub.C 62.9) were then determined using the
.sup.1H-.sup.13C HSQC-DEPT correlations to complete the assignment
of Glc.sub.IV.
[0559] A summary of the .sup.1H and .sup.13C chemical shifts for
the glycoside at C-13 are shown in the table below:
TABLE-US-00057 .sup.1H and .sup.13C NMR (500 and 125 MHz,
pyridine-d.sub.5), Assignments of the Reb D2 C-13 glycoside. Reb D2
Position .sup.13C .sup.1H Glc.sub.II-1 98.7 5.07* Glc.sub.II-2 81.3
4.37 m Glc.sub.II-3 88.4 4.18 t (9.0) Glc.sub.II-4 71.1 3.88 t
(8.6) Glc.sub.II-5 77.9 3.79 m Glc.sub.II-6 63.2 4.08 m 4.46 m
Glc.sub.III-1 105.3 5.57 d (7.6) Glc.sub.III-2 76.8 4.19 m
Glc.sub.III-3 78.9 4.24 m Glc.sub.III-4 72.4 4.27 m Glc.sub.III-5
78.8 3.94 m Glc.sub.III-6 63.5 4.41 m 4.50 m Glc.sub.IV-1.sup.
105.3 5.34 d (7.9) Glc.sub.IV-2.sup. 75.8 4.06 m Glc.sub.IV-3.sup.
78.9.sup.# 4.22 m.sup.# Glc.sub.IV-4.sup. 72.0 4.18 m
Glc.sub.IV-5.sup. 79.3 4.10 m Glc.sub.IV-6.sup. 62.9 4.32 m 4.58
m
[0560] A summary of the key HMBC, COSY, and 1D-TOCSY correlations
used to assign the C-13 glycoside region are provided below:
##STR00011##
[0561] NMR and MS analyses allowed a full assignment of structure,
shown below. The chemical name of the compound is
13-[(2-O-.beta.-D-glucopyranosyl-3-O-.beta.-D-glucopyranosyl-.beta.-D-glu-
copyranosyl)oxy] ent-kaur-16-en-19-oic
acid-[(6-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl) ester]
(rebaudioside D2 or reb D2). The compound is an isomer of
rebaudioside D.
##STR00012##
Example 40
[0562] Isolation and Characterization of Reb M2
[0563] Crude Reaction Sample.
[0564] The sample, Lot CB-2977-106, used for isolation was prepared
according to Example 22 with UGTSL (GI #460409128).
[0565] HPLC Analysis.
[0566] Preliminary HPLC analyses was performed using a Waters 2695
Alliance System with the following method: Phenomenex Synergi
Hydro-RP, 4.6.times.250 mm, 4 .mu.m (p/n 00G-4375-E0); Column Temp:
55.degree. C.; Mobile Phase A: 0.0284% NH.sub.4OAc and 0.0116% HOAc
in water; Mobile Phase B: Acetonitrile (MeCN); Flow Rate: 1.0
mL/min; Injection volume: 10 .mu.L. Detection was by UV (210 nm)
and CAD.
[0567] Gradient:
TABLE-US-00058 Time (min) % A % B 0.0-5.0 100 0 20 20 80 25 20 80
30 100 0
[0568] Analyses of semi-preparative purification fractions were
performed with the following method: Waters Atlantis dC18,
4.6.times.100 mm, 5 .mu.m (p/n 186001340); Mobile Phase A: 25% MeCN
in water; Mobile Phase B: 30% MeCN in water; Flow Rate: 1.0 mL/min;
Injection volume: 10 .mu.L. Detection was by CAD.
[0569] Gradient:
TABLE-US-00059 Time (min) % A % B 0.0-8.5 75 25 10.0 71 29 16.5 70
30 18.5-24.5 66 34 26.5-29.0 48 52 31-37 30 70 38 75 25
[0570] LC-MS.
[0571] Preliminary analysis of the semi-synthetic steviol glycoside
mixture was carried out on a Waters AutoPurification HPLC/MS System
with a Waters 3100 Mass Detector operating in negative ion mode.
Analysis of the sample was performed using the following method:
Phenomenex Synergi Hydro-RP, 4.6.times.250 mm, 4 .mu.m (p/n
00G-4375-E0); Column Temp: 55.degree. C.; Mobile Phase A: 0.0284%
NH.sub.4OAc and 0.0116% HOAc in water; Mobile Phase B: MeCN; Flow
Rate: 1.0 mL/min; Injection volume: 10 .mu.L. Detection was by UV
(210 nm), and MSD (-ESI m/z 500-2000). Gradient conditions were as
listed above.
[0572] Isolation by HPLC.
[0573] The purification was performed in two steps. The first
method used for the semi-preparative purification is summarized
below. Column: Waters Atlantis dC18, 30.times.100 mm, 5 (p/n
186001375); Mobile Phase A: 25% MeCN in water; Mobile Phase B: 30%
MeCN in water; Flow Rate: 45 mL/min; Injection load: 160 mg
dissolved in 20 mL of water. Detection was by UV (205 nm).
[0574] Gradient:
TABLE-US-00060 Time (min) % A % B 0.0-5.0 100 0 20 20 80 25 20 80
30 100 0
[0575] The secondary purification used the same column and
conditions, but isocratic mobile phase: 20% MeCN in water.
[0576] MS and MS/MS.
[0577] MS and MS/MS data were generated with a Waters QT of Premier
mass spectrometer equipped with an electrospray ionization source.
Samples were analyzed by negative ESI. Samples were diluted with
H.sub.2O:MeCN (1:1) by 50 fold and introduced via infusion using
the onboard syringe pump. The samples were diluted to yield good
s/n which occurred at an approximate concentration of 0.01
mg/mL.
[0578] NMR.
[0579] The sample was prepared by dissolving .about.1.0 mg in 150
.mu.L of D.sub.2O and NMR data were acquired on a Bruker Avance 500
MHz instrument with a 2.5 mm inverse detection probe. The .sup.1H
NMR and .sup.13C NMR spectra were referenced to the residual
solvent signal HDO (.delta..sub.H 4.79 ppm) and TSP (.delta..sub.C
0.00 ppm), respectively.
[0580] Results and Discussion
[0581] Isolation and Purification.
[0582] Isolation was performed using on a steviol glycoside
mixture, Lot number CB-2977-106, prepared according to Example 22
with UGTSL (GI #460409128). The material was analyzed by LC-MS
using the method described above (FIG. 11). The targeted peak of
interest was that at 4.1 min in the TIC chromatogram. The mass
spectrum of this peak provided a [M-H].sup.- ion at m/z 1289.7. The
provided sample was preliminarily processed in a single injection
(160 mg) using the first method condition provided above. This
method fractionated the material into `polar` and `non-polar`
mixtures of glycosides. The `polar` mixture was then reprocessed
using the second-step conditions provided above. The
semi-preparative HPLC trace is shown in FIG. 12. From this
semi-preparative collection, the peak was isolated with a purity
>99% (CAD, AUC). The fraction analysis is provided in FIG. 13.
Following the purification, the fractions were concentrated by
rotary evaporation at 35.degree. C. and lyophilized. Approximately
1 mg was obtained.
[0583] Mass Spectrometry.
[0584] The ESI-TOF mass spectrum acquired by infusing a sample of
CC-00300 showed a [M-H].sup.- ion at m/z 1289.5266. The mass of the
[M-H].sup.- ion was in good agreement with the molecular formula
C.sub.56H.sub.90O.sub.33 (calcd for C.sub.56H.sub.89O.sub.33:
1289.5286, error: -1.6 ppm) expected for reb M2. The MS data
confirmed that CC-00300 has a nominal mass of 1290 Daltons with the
molecular formula, C.sub.56H.sub.90O.sub.33.
[0585] The MS/MS spectrum (selecting the [M-H].sup.- ion at m/z
1289.5 for fragmentation) indicated the loss of three glucose units
at m/z 803.3688 and sequential loss of three glucose moieties at
m/z 641.3165, 479.2633 and 317.2082.
[0586] NMR Spectroscopy.
[0587] A series of NMR experiments including .sup.1H NMR (FIG. 21),
.sup.13C NMR (FIGS. 22 and 23), .sup.1H-.sup.1H COSY (FIG. 24),
HSQC-DEPT (FIG. 25), HMBC (FIGS. 26 and 27), and 1D-TOCSY were
performed to allow assignment of reb M2.
[0588] The .sup.1H, .sup.1H-.sup.1H COSY, .sup.1H-.sup.13C
HSQC-DEPT and .sup.1H-.sup.13C HMBC NMR data indicated that the
central core of the glycoside is a diterpene. The presence of six
anomeric protons observed in the .sup.1H and .sup.1H-.sup.13C
HSQC-DEPT spectra confirm six sugar units in the structure. The
methylene .sup.13C resonance at .delta..sub.C 70.9 in the
.sup.1H-.sup.13C HSQC-DEPT spectrum indicated the presence of a
1.fwdarw.6 sugar linkage in the structure. The linkages of sugar
units were assigned using .sup.1H-.sup.13C HMBC and 1D-TOCSY
correlations.
[0589] A HMBC correlation from the methyl protons at .delta..sub.H
1.29 to the carbonyl at .delta..sub.C 181.5 allowed assignment of
one of the tertiary methyl groups (C-18) as well as C-19 and
provided a starting point for the assignment of the rest of the
aglycone. Additional HMBC correlations from the methyl protons
(H-18) to carbons at .delta..sub.C 39.8, 43.7, and 59.2 allowed
assignment of C3, C4, and C5. Analysis of the .sup.1H-.sup.13C
HSQC-DEPT data indicated that the carbon at .delta..sub.C 39.8 was
a methylene group and the carbon at .delta..sub.C 59.2 was a
methine which were assigned as C-3 and C-5, respectively. This left
the carbon at .delta..sub.C 43.7, which did not show a correlation
in the HSQC-DEPT spectrum, to be assigned as the quaternary carbon,
C-4. The .sup.1H chemical shifts for C-3 (.delta..sub.H 1.16 and
2.28) and C-5 (.delta..sub.H 1.24) were assigned using the
HSQC-DEPT data. A COSY correlation between one of the H-3 protons
O.sub.H 1.16) and a proton at .delta..sub.H 1.49 allowed assignment
of one of the H-2 protons which in turn showed a correlation with a
proton at .delta..sub.H 0.92 which was assigned to C-1. The
remaining .sup.1H and .sup.13C chemical shifts for C-1 and C-2 were
then assigned on the basis of additional COSY and HSQC-DEPT
correlations and are summarized in the table below.
TABLE-US-00061 .sup.1H NMR (500 MHz, D.sub.2O) and .sup.13C NMR
(125 MHz, D.sub.2O/TSP) Assignments of the Reb M2 aglycone.
Position .sup.13C .sup.1H 1 41.9 0.92 m 1.93 m 2 21.8 1.49 m 1.86 m
3 39.8 1.16 m 2.28 d (13.4) 4 43.7 -- 5 59.2 1.24 d (12.1) 6 24.4
1.73 m 1.94 m 7 44.2 1.49 m 1.56 m 8 46.9 -- 9 55.5 1.09 d (7.7) 10
42.4 -- 11 22.6 1.66 m 1.70 m 12 39.9 1.60 m 2.00 m 13 90.9 -- 14
46.9 1.53 d (12.6) 2.21 d (13.6) 15 49.4 2.15 d (17.2) 2.18 d
(18.1) 16 164.0 -- 17 107.0 4.98 s 5.16 s 18 31.0 1.29 s 19 181.5
-- 20 19.1 0.92 s
[0590] The other tertiary methyl singlet, observed at .delta..sub.H
0.92 showed HMBC correlations to C-1 and C-5 and was assigned as
C-20. The methyl protons showed additional HMBC correlations to a
quaternary carbon (.delta..sub.C 42.4) and a methine (.delta..sub.C
55.5) which were assigned as C-10 and C-9, respectively. COSY
correlations between H-5 (.delta..sub.H 1.24) and protons at
.delta..sub.H 1.73 and 1.94 then allowed assignment of the H-6
protons which in turn showed correlations to protons at
.delta..sub.H 1.49 and 1.56 which were assigned to C-7. The
.sup.13C chemical shifts for C-6 (.delta..sub.C 24.4) and C-7
(.delta..sub.C 44.2) were then determined from the HSQC-DEPT data.
COSY correlations between H-9 (.delta..sub.H 1.09) and protons at
.delta..sub.H 1.66 and 1.70 allowed assignment of the H-11 protons
which in turn showed COSY correlations to protons at .delta..sub.H
1.60 and 2.00 which were assigned as the H-12 protons. The
HSQC-DEPT data was then used to assign C-11 (.delta..sub.C 22.6)
and C-12 (.delta..sub.C 39.9). The olefinic protons observed at
.delta..sub.H 4.98 and 5.16 showed HMBC correlations to C-13
(.delta..sub.C 90.9) and were assigned to C-17 (.delta..sub.C 107.0
via HSQC-DEPT). The olefinic protons H-17 showed HMBC correlations
to a carbon at .delta..sub.C 49.4 which was assigned as C-15. An
additional HMBC correlation from H-9 to a methylene carbon at
.delta..sub.C 46.9 then allowed assignment of C-14. The .sup.1H
chemical shifts at C-14 (.delta..sub.H 1.53 and 2.21) and C-15
(.delta..sub.H 2.15 and 2.18) were assigned using the HSQC-DEPT
data.
[0591] A summary of the key HMBC and COSY correlations used to
assign the aglycone region are provided below:
##STR00013##
[0592] Analysis of the .sup.1H-.sup.13C HSQC-DEPT data confirmed
the presence of six anomeric protons. Three of the anomeric protons
were well resolved at .delta..sub.H 5.65 (.delta..sub.C 95.5), 4.92
(.delta..sub.C 104.9), and 4.50 (.delta..sub.C 105.7) in the
.sup.1H NMR spectrum. The remaining three anomeric protons observed
at .delta..sub.H 4.85 (.delta..sub.C 98.4), 4.84 (.delta..sub.C
105.0), and 4.83 (.delta..sub.C 105.3) were overlapped by the
residual solvent resonance in the .sup.1H spectrum. The anomeric
proton observed at .delta..sub.H 5.65 showed a HMBC correlation to
C-19 which indicated that it corresponds to the anomeric proton of
Glc.sub.I. Similarly, the anomeric proton observed at .delta..sub.H
4.85 showed a HMBC correlation to C-13 allowing it to be assigned
as the anomeric proton of Glc.sub.II.
[0593] The Glc.sub.I anomeric proton (.delta..sub.H 5.65) showed a
COSY correlation to a proton at .delta..sub.H 3.96 which was
assigned as Glc.sub.I H-2 which in turn showed a COSY correlation
to a proton at .delta..sub.H 3.89 (Glc.sub.I H-3) which showed a
COSY correlation with a proton at .delta..sub.H 3.71 (Glc.sub.I
H-4). Due to data overlap, the COSY spectrum did not allow
assignment of the H-5 or H-6 protons. Therefore, a series of
1D-TOCSY experiments were performed using selective irradiation of
the Glc.sub.I anomeric proton with several different mixing times.
In addition to confirming the assignments for Glc.sub.I H-2 through
H-4, the 1D-TOCSY data showed a proton at .delta..sub.H 3.73
assigned as Glc.sub.I H-5 and a proton at .delta..sub.H 4.15
assigned as one of the Glc.sub.I H-6 protons. The latter proton was
also used for 1D-TOCSY experiments. The selective irradiation of
H-6 with several different mixing times also confirmed the
assignment of Glc.sub.I H-1 to H-5 as well as the remaining
methylene proton of H-6 (.delta..sub.H 4.00). Assignment of the
.sup.13C chemical shifts for Glc.sub.I C-2 (.delta..sub.C 80.5),
C-3 (.delta..sub.C 79.0), C-4 (.delta..sub.C 71.5), C-5
(.delta..sub.C 79.0), and C-6 (.delta..sub.C 70.9) was determined
using the .sup.1H-.sup.13C HSQC-DEPT data to complete the
assignment of Glc.sub.I. Furthermore, the presence of a methylene
.sup.13C resonance at .delta..sub.C 70.9 in the .sup.1H-.sup.13C
HSQC-DEPT spectrum indicated a 1.fwdarw.6 sugar linkage of
Glc.sub.I in the structure.
[0594] Two of the unassigned glucose moieties were assigned as
substituents at C-2 and C-6 of Glc.sub.I on the basis of HMBC
correlations. The anomeric proton observed at .delta..sub.H 4.83
showed an HMBC correlation to Glc.sub.I C-2 and was assigned as the
anomeric proton of Glc.sub.V. The anomeric proton observed at
.delta..sub.H 4.50 showed a HMBC correlation to Glc.sub.I C-6 and
was assigned as the anomeric proton of Glc.sub.VI. The reciprocal
HMBC correlations from Glc.sub.I H-2 to the anomeric carbon of
Glc.sub.V and from Glc.sub.I H-6 to the anomeric carbon of
Glc.sub.VI were also observed.
[0595] The anomeric proton of Glc.sub.V (.delta..sub.H 4.83) showed
a COSY correlation with a proton at .delta..sub.H 3.32 which was
assigned as Glc.sub.V H-2. The Glc.sub.V H-2 in turn showed a COSY
correlation to a proton at .delta..sub.H 3.51 (Glc.sub.V H-3). This
latter proton showed an additional correlation with a proton at
.delta..sub.H 3.38 (Glc.sub.V H-4). H-4 also showed a COSY
correlation to a proton at .delta..sub.H 3.55 (Glc.sub.V H-5) and
Glc.sub.V H-5 in turn showed a COSY correlation to Glc.sub.V H-6
protons (.delta..sub.H 3.76 and 3.97). Assignment of the .sup.13C
chemical shifts for Glc.sub.V C-2 (.delta..sub.C 78.5), C-3
(.delta..sub.C 78.7), C-4 (.delta..sub.C 72.9), C-5 (.delta..sub.C
78.8), and C-6 (.delta..sub.C 63.6) was determined using the
HSQC-DEPT data. HMBC correlations from Glc.sub.V H-3 to C-2 and C-4
and also from Glc.sub.V H-4 to C-3 and C-6 confirmed the
assignments made above to complete the assignment of Glc.sub.V.
[0596] Another glucose moiety was assigned as a substituent at C-6
of Glc.sub.I on the basis of .sup.1H-.sup.13C HSQC-DEPT and HMBC
correlations. The relatively downfield shift of a methylene
.sup.13C resonance of Glc.sub.I at .delta..sub.C 70.9 in the
HSQC-DEPT spectrum indicated a 1.fwdarw.6 sugar linkage of
Glc.sub.I. The anomeric proton observed at .delta..sub.H 4.50
showed a HMBC correlation to Glc.sub.I C-6 and was assigned as the
anomeric proton of Glc.sub.VI. Similarly, methylene protons of
Glc.sub.I showed HMBC correlations to the anomeric carbon of
Glc.sub.VI and this confirmed the presence of a 1.fwdarw.6 sugar
linkage between Glc.sub.I and Glc.sub.VI. The Glc.sub.VI anomeric
proton showed a COSY correlation to a proton at .delta..sub.H 3.33
which was assigned as Glc.sub.VI H-2 which in turn showed a COSY
correlation to a proton at .delta..sub.H 3.49 (Glc.sub.VI H-3). Due
to data overlap, the COSY spectrum did not allow assignment of
Glc.sub.V H-4 to H-6 based on the COSY correlations. Therefore, a
series of 1D-TOCSY experiments were performed using selective
irradiation of the Glc.sub.VI anomeric proton with different mixing
times. In addition to confirming the assignments for Glc.sub.VI H-2
through H-3, the 1D-TOCSY data showed protons at .delta..sub.H 3.45
(Glc.sub.VI H-4) and .delta..sub.H 3.48 (Glc.sub.VI H-5) and
protons at .delta..sub.H 3.92 and 3.94 assigned for Glc.sub.VI H-6
protons. Assignment of the .sup.13C chemical shifts for Glc.sub.VI
C-2 (.delta..sub.C 78.1), C-3 (.delta..sub.C 78.6), C-4
(.delta..sub.C 72.3), C-5 (.delta..sub.C 78.8), and C-6
(.delta..sub.C 64.1) was determined using the .sup.1H-.sup.13C
HSQC-DEPT data to complete the assignment of Glc.sub.VI.
[0597] A summary of the .sup.1H and .sup.13C chemical shifts for
the glycoside at C-19 are found in the table below:
TABLE-US-00062 H NMR (500 MHz, D.sub.2O) and .sup.13C NMR (125 MHz,
D.sub.2O/TSP) Assignments of the Reb M2 glycoside. Position
.sup.13C .sup.1H Glc.sub.I-1 95.5 5.65 d (7.6) Glc.sub.I-2 80.5
3.96 m Glc.sub.I-3 79.0 3.89 m Glc.sub.I-4 71.5 3.71 m Glc.sub.I-5
79.0 3.73 m Glc.sub.I-6 70.9 4.00 m 4.15 d (11.7) Glc.sub.V-1
105.3* 4.83* d (8.0) Glc.sub.V-2 78.5 3.32 m Glc.sub.V-3 78.7 3.51
m Glc.sub.V-4 72.9 3.38 m Glc.sub.V-5 78.8 3.55 m Glc.sub.V-6 63.6
3.76 m 3.97 m Glc.sub.VI-1 105.7 4.50 d (7.9) Glc.sub.VI-2 78.1
3.33 m Glc.sub.VI-3 78.6 3.49 m Glc.sub.VI-4 72.3 3.45 m
Glc.sub.VI-5 78.8 3.48 m Glc.sub.VI-6 64.1 3.92 m 3.94 m *.sup.1H
and .sup.13C values can be exchangeable with Glc.sub.VI-1 of the
following table.
[0598] A summary of the key HMBC, COSY, and 1D-TOCSY correlations
used to assign the C-19 glycoside region are provided below:
TABLE-US-00063 .sup.1H NMR (500 MHz, D.sub.2O) and .sup.13C NMR
(125 MHz, D.sub.2O/TSP) Assignments of the Reb M2 glycoside.
Position .sup.13C.sup.# .sup.1H Glc.sub.II-1 98.4 4.85 d (7.8)
Glc.sub.II-2 81.7 3.75 m Glc.sub.II-3 88.0 3.98 m Glc.sub.II-4 71.3
3.54 m Glc.sub.II-5 80.5 3.96 m Glc.sub.II-6 63.6 3.45 m 3.77 m
Glc.sub.III-1 104.9 4.92 d (7.9) Glc.sub.III-2 76.3 3.32 m
Glc.sub.III-3 78.8 3.51 m Glc.sub.III-4 73.3 3.26 t (9.5)
Glc.sub.III-5 78.8 3.44 m Glc.sub.III-6 64.4 3.75 m 3.94 m
Glc.sub.IV-1.sup. 105.0 4.84 d (7.8) Glc.sub.IV-2.sup. 76.1 3.41 m
Glc.sub.IV-3.sup. 78.8 3.46 m Glc.sub.IV-4.sup. 72.5 3.45 m
Glc.sub.IV-5.sup. 81.7 3.75 m Glc.sub.IV-6.sup. 65.8 3.55 m 3.78
m
[0599] Assignment of Glc.sub.II was carried out in a similar
manner. The Glc.sub.II anomeric proton (.delta..sub.H 4.85) showed
a COSY correlation to a proton at .delta..sub.H 3.75 which was
assigned as Glc.sub.II H-2 which in turn showed a COSY correlation
to a proton at .delta..sub.H 3.98 (Glc.sub.II H-3). This latter
proton showed an additional correlation with a proton at
.delta..sub.H 3.54 (Glc.sub.II H-4). H-4 also showed a COSY
correlation to a proton at .delta..sub.H 3.96 (Glc.sub.II H-5).
Glc.sub.II H-5 also showed a COSY correlation to Glc.sub.II H-6
protons (.delta..sub.H 3.77 and 3.45). Assignment of the .sup.13C
chemical shifts for Glc.sub.II C-2 (.delta..sub.C 81.7), C-3
(.delta..sub.C 88.0), C-4 (.delta..sub.C 71.3), C-5 (.delta..sub.C
80.5), and C-6 (.delta..sub.C 63.6) was determined using the
HSQC-DEPT data. HMBC correlations from Glc.sub.II H-3 to C-2 and
C-4 and also from Glc.sub.II H-4 to C-3 and C-6 confirmed the
assignments made above to complete the assignment of
Glc.sub.II.
[0600] Two of the remaining unassigned glucose moieties were
assigned as substituents at C-2 and C-3 of Glc.sub.II on the basis
of HMBC correlations. The anomeric proton observed at .delta..sub.H
4.92 showed a HMBC correlation to Glc.sub.II C-2 and was assigned
as the anomeric proton of Glc.sub.III. The anomeric proton observed
at .delta..sub.H 4.84 showed a HMBC correlation to Glc.sub.II C-3
and was assigned as the anomeric proton of Glc.sub.IV. The
reciprocal HMBC correlations between Glc.sub.II H-2 and the
anomeric carbon of Glc.sub.III and between Glc.sub.II H-3 and the
anomeric carbon of Glc.sub.IV were also observed.
[0601] The anomeric proton of Glc.sub.III (.delta..sub.H 4.92)
showed a COSY correlation with a proton at .delta..sub.H 3.32 which
was assigned as Glc.sub.III H-2. Due to data overlap, the COSY
spectrum did not allow assignment of H-3 to H-6 protons. Therefore,
a series of 1D-TOCSY experiments were performed using selective
irradiation of the Glc.sub.III anomeric proton with different
mixing times. In addition to confirming the assignments for
Glc.sub.III H-2, the 1D-TOCSY data showed protons at .delta..sub.H
3.51 (Glc.sub.III H-3), .delta..sub.H 3.26 (Glc.sub.III H-4), and
.delta..sub.H 3.44 (Glc.sub.III H-5). Once H-4 was assigned using
1D-TOCSY data, COSY correlations from H-4 to H-5 and in turn to H-6
were used to assign H-6. In the COSY spectrum, Glc.sub.III H-4
showed a correlation to Glc.sub.III H-5, which in turn showed COSY
correlations to .delta..sub.H 3.94 and 3.75 of Glc.sub.III H-6a and
H-6b, respectively. The .sup.13C chemical shifts for Glc.sub.III
C-2 (.delta..sub.C 76.3), C-3 (.delta..sub.C 78.8), C-4
(.delta..sub.C 73.3), C-5 (.delta..sub.C 78.8), and C-6
(.delta..sub.C 64.4) were then determined using the
.sup.1H-.sup.13C HSQC-DEPT correlations to complete the assignment
of Glc.sub.III.
[0602] The anomeric proton of Glc.sub.IV (.delta..sub.H 4.84) which
showed a COSY correlation to a proton at .delta..sub.H 3.41 was
assigned as Glc.sub.II/H-2 which in turn showed a COSY correlation
to a proton at .delta..sub.H 3.46 (Glc.sub.IV H-3). This latter
proton showed an additional correlation with a proton at
.delta..sub.H 3.45 (Glc.sub.II, H-4) which also showed a COSY
correlation to a proton at .delta..sub.H 3.75 (Glc.sub.IV H-5).
Glc.sub.IV H-5 also showed a COSY correlation to Glc.sub.IV H-6
protons (.delta..sub.H 3.55 and 3.78). Assignment of the .sup.13C
chemical shifts for Glc.sub.IV C-2 (.delta..sub.C 76.1), C-3
(.delta..sub.C 78.8), C-4 (.delta..sub.C 72.5), C-5 (.delta..sub.C
81.7), and C-6 (.delta..sub.C 65.8) was determined using the
HSQC-DEPT data. HMBC correlations from Glc.sub.IV H-3 to C-4 and
C-5 and also from Glc.sub.II, H-4 to C-3 and C-6 confirmed the
assignments made above to complete the assignment of
Glc.sub.IV.
[0603] A summary of the .sup.1H and .sup.13C chemical shifts for
the glycoside at C-13 are found in the following table:
TABLE-US-00064 .sup.1H NMR (500 MHz, D.sub.2O) and .sup.13C NMR
(125 MHz, D.sub.2O/TSP) Assignments of the Reb M2 glycoside.
Position .sup.13C.sup.# .sup.1H Glc.sub.II-1 98.4 4.85 d (7.8)
Glc.sub.II-2 81.7 3.75 m Glc.sub.II-3 88.0 3.98 m Glc.sub.II-4 71.3
3.54 m Glc.sub.II-5 80.5 3.96 m Glc.sub.II-6 63.6 3.45 m 3.77 m
Glc.sub.III-1 104.9 4.92 d (7.9) Glc.sub.III-2 76.3 3.32 m
Glc.sub.III-3 78.8 3.51 m Glc.sub.III-4 73.3 3.26 t (9.5)
Glc.sub.III-5 78.8 3.44 m Glc.sub.III-6 64.4 3.75 m 3.94 m
Glc.sub.IV-1.sup. 105.0 4.84 d (7.8) Glc.sub.IV-2.sup. 76.1 3.41 m
Glc.sub.IV-3.sup. 78.8 3.46 m Glc.sub.IV-4.sup. 72.5 3.45 m
Glc.sub.IV-5.sup. 81.7 3.75 m Glc.sub.IV-6.sup. 65.8 3.55 m 3.78
m
[0604] A summary of the key HMBC, COSY, and 1D-TOCSY correlations
used to assign the C-13 glycoside region are provided below:
##STR00014##
[0605] NMR and MS analyses allowed a full assignment of its
structure, shown below. The chemical name of the compound is
13-[(2-O-.beta.-D-glucopyranosyl-3-O-13-D-glucopyranosyl-.beta.-D-glucopy-
ranosyl)oxy] ent-kaur-16-en-19-oic
acid-[(2-O-.beta.-D-glucopyranosyl-6-O-.beta.-D-glucopyranosyl-.beta.-D-g-
lucopyranosyl) ester] (rebaudioside M2 or reb M2). The compound is
an isomer of rebaudioside M.
##STR00015##
Example 41
[0606] Directed evolution of UGT76G1 for the conversion of
Rebaudioside D to Rebaudioside X (Round 2)
[0607] The most active clone from the first round of directed
evolution of UGT76G1 (see EXAMPLE 26 UGT76G1var94 containing
mutations: Q266E.sub.-- P272A_R334K_G348P.sub.-- L379G) was chosen
as baseline clone for round 2. A list of 53 mutations was
established containing different identified positive mutations from
the first round and new mutations obtained by DNA2.0 ProteinGPStm
strategy. This list of mutations was subsequently used to design 92
variant genes that contained each 3 different mutations. After
codon-optimized for expression in E. coli the genes were
synthesized, subcloned in the pET30a+ plasmid and used for
transformation of E. coli BL21 (DE3) chemically competent cells.
The obtained cells were grown in Petri-dishes on solid LB medium in
the presence of Kanamycin. Suitable colonies were selected and
allowed to grow in liquid LB medium in tubes. Glycerol was added to
the suspension as cryoprotectant and 400 .mu.L aliquots were stored
at -20.degree. C. and at -80.degree. C.
[0608] These storage aliquots of E. coli BL21(DE3) containing the
pET30a+_UGT76G1var plasmids were thawed and added to LBGKP medium
(20 g/L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM
Phosphate buffer pH 7.00; 2.5 g/L glucose and 50 mg/L of
Kanamycine). This culture was allowed to shake in a 96 microtiter
plate at 30.degree. C. for 8 h.
[0609] 3.95 mL of production medium containing 60 g/L of Overnight
Express.TM. Instant TB medium (Novagen.RTM.), 10 g/L of glycerol
and 50 mg/L of Kanamycin was inoculated with 50 .mu.L of above
described culture. In a 48 deepwell plate the resulting culture was
allowed to stir at 20.degree. C. The cultures gave significant
growth and a good OD (600 nm) was obtained. After 44 h, the cells
were harvested by centrifugation and frozen.
[0610] Lysis was performed by addition of Bugbuster.RTM. Master mix
(Novagen.RTM.) to the thawed cells and the lysate was recovered by
centrifugation. Activity tests were performed with 100 .mu.L of
fresh lysate that was added to a solution of Rebaudioside D (final
concentration 0.5 mM), MgCl.sub.2 (final concentration 3 mM) and
UDP-Glucose (final concentration 2.5 mM) in 50 mM phosphate buffer
pH 7.2.
[0611] The reaction was allowed to run at 30.degree. C. and samples
were taken after 2, 4, 7 and 24 h. to determine conversion and
initial rate by HPLC (CAD detection) using the analytical method
that was described above for the transformation of Rebaudioside D
to Rebaudioside X. In parallel the experiments were performed with
baseline clone, Round1-Var94. The conversion after 22 h. and
initial rate for this baseline clone was defined as 100% and the
normalized conversions and initial rates for the round 2 clones are
depicted in the following table:
TABLE-US-00065 Normalized conversion Normalized initial Clone
Mutations* Reb D to Reb X after 22 h. rate (0-4 h) Round1-Var94
UGT76G1 100% 100% (Q266E_P272A_R334K_G348P_L379G) baseline clone
Round2-Var1 Round1-Var94 (A213N_P348G_I411V) 70% 86% Round2-Var2
Round1-Var94 (K303G_I423M_Q425E) 120% 134% Round2-Var3 Round1-Var94
(V20L_N138K_S147G) 14% 15% Round2-Var4 Round1-Var94
(I16V_V133A_L299I) 37% 43% Round2-Var5 Round1-Var94
(S241V_S274G_Q432E) 75% 72% Round2-Var6 Round1-Var94
(I16V_L139V_I218V) 62% 68% Round2-Var7 Round1-Var94
(K334R_N409K_Q432E) 104% 92% Round2-Var8 Round1-Var94
(I15L_R141T_I407V) 17% 26% Round2-Var9 Round1-Var94
(R141T_K303G_G379L) 31% 42% Round2-Var10 Round1-Var94
(I190L_K303G_P348G) 131% 149% Round2-Var11 Round1-Var94
(E266Q_F314S_N409R) 106% 132% Round2-Var12 Round1-Var94
(V133A_I295V_K303G) 43% 49% Round2-Var13 Round1-Var94
(I16V_S241V_N409R) 80% 79% Round2-Var14 Round1-Var94
(A239V_K334R_G379L) 58% 55% Round2-Var15 Round1-Var94
(I190L_K393R_V396L) 118% 126% Round2-Var16 Round1-Var94
(L101F_I295M_K393R) 84% 89% Round2-Var17 Round1-Var94
(A239V_E266Q_Q425E) 96% 101% Round2-Var18 Round1-Var94
(V20L_I190L_I423M) 98% 98% Round2-Var19 Round1-Var94
(V20L_G379L_S456L) 84% 81% Round2-Var20 Round1-Var94
(K334R_P348G_N409R) 73% 73% Round2-Var21 Round1-Var94
(E231A_S241V_E449D) 53% 50% Round2-Var22 Round1-Var94
(K188R_L299I_V394I) 56% 59% Round2-Var23 Round1-Var94
(E231A_S274G_V394I) 110% 124% Round2-Var24 Round1-Var94
(S42A_I295V_Q432E) 71% 78% Round2-Var25 Round1-Var94
(A213N_A272P_K334R) 95% 80% Round2-Var26 Round1-Var94
(L158Y_S274K_N409K) 80% 50% Round2-Var27 Round1-Var94
(K188R_I295M_Q425E) 132% 116% Round2-Var28 Round1-Var94
(I15L_I295M_V394I) 53% 36% Round2-Var29 Round1-Var94
(V133A_A239V_V394I) 47% 30% Round2-Var30 Round1-Var94
(L158Y_F314S_K316R) 107% 72% Round2-Var31 Round1-Var94
(L158Y_A239V_A272P) 54% 30% Round2-Var32 Round1-Var94
(F46I_D301N_V396L) 109% 101% Round2-Var33 Round1-Var94
(L101F_I218V_Q432E) 78% 54% Round2-Var34 Round1-Var94
(I16V_F46I_I295M) 110% 95% Round2-Var35 Round1-Var94
(A213N_E266S_I407V) 98% 79% Round2-Var36 Round1-Var94
(A239V_S274K_I295M) 102% 89% Round2-Var37 Round1-Var94
(A239V_F314S_S450K) 105% 99% Round2-Var38 Round1-Var94
(L139V_K188R_D301N) 66% 51% Round2-Var39 Round1-Var94
(I45V_I218V_S274K) 87% 58% Round2-Var40 Round1-Var94
(S241V_K303G_V394I) 78% 57% Round2-Var41 Round1-Var94
(R141T_S274G_K334R) 41% 28% Round2-Var42 Round1-Var94
(V217L_S274G_L299I) 47% 34% Round2-Var43 Round1-Var94
(S274G_D301N_P348G) 98% 91% Round2-Var44 Round1-Var94
(E231A_N409R_S450K) 87% 65% Round2-Var45 Round1-Var94
(R64H_E231A_K316R) 88% 64% Round2-Var46 Round1-Var94
(V394I_N409K_I411V) 110% 100% Round2-Var47 Round1-Var94
(I45V_I295M_K303G) 113% 88% Round2-Var48 Round1-Var94
(L101F_V396L_L398V) 46% 43% Round2-Var49 Round1-Var94
(N27S_L101F_S447A) 54% 37% Round2-Var50 Round1-Var94
(S274G_F314S_L398V) 129% 156% Round2-Var51 Round1-Var94
(E266Q_L299I_K393R) 70% 51% Round2-Var52 Round1-Var94
(V217L_E266S_V394I) 62% 48% Round2-Var53 Round1-Var94
(N138K_A272P_N409R) 118% 102% Round2-Var54 Round1-Var94
(E266S_F314S_Q432E) 124% 146% Round2-Var55 Round1-Var94
(D301N_G379L_L398V) 56% 45% Round2-Var56 Round1-Var94
(F46I_E266S_K334R) 123% 142% Round2-Var57 Round1-Var94
(A272P_V394I_Q432E) 133% 142% Round2-Var58 Round1-Var94
(V394I_I407V_S456L) 118% 114% Round2-Var59 Round1-Var94
(I218V_E266Q_I423M) 106% 98% Round2-Var60 Round1-Var94
(A272P_G379L_I407V) 80% 63% Round2-Var61 Round1-Var94
(E231A_K303G_S456L) 113% 110% Round2-Var62 Round1-Var94
(I190L_E266Q_I407V) 150% 167% Round2-Var63 Round1-Var94
(N27S_L139V_I295V) 43% 25% Round2-Var64 Round1-Var94
(V217L_I423M_S447A) 67% 51% Round2-Var65 Round1-Var94
(L158Y_E266S_E449D) 68% 43% Round2-Var66 Round1-Var94
(S42A_F46I_I407V) 160% 203% Round2-Var67 Round1-Var94
(N138K_E231A_D301N) 118% 93% Round2-Var68 Round1-Var94
(K188R_G379L_N409R) 52% 35% Round2-Var69 Round1-Var94
(I15L_E231A_V396L) 38% 22% Round2-Var70 Round1-Var94
(E231A_Q425E_Q432E) 115% 119% Round2-Var71 Round1-Var94
(D301N_K316R_Q425E) 126% 121% Round2-Var72 Round1-Var94
(L139V_I295M_F314S) 76% 91% Round2-Var73 Round1-Var94
(S147G_E266S_D301N) 30% 18% Round2-Var74 Round1-Var94
(R64H_S147G_S447A) 23% 12% Round2-Var75 Round1-Var94
(S42A_K303G_L398V) 95% 110% Round2-Var76 Round1-Var94
(I45V_D301N_E449D) 62% 60% Round2-Var77 Round1-Var94
(V133A_E266S_I411V) 37% 28% Round2-Var78 Round1-Var94
(I45V_N409R_Q425E) 63% 59% Round2-Var79 Round1-Var94
(R141T_A272P_F314S) 23% 10% Round2-Var80 Round1-Var94
(E266S_S274G_N409R) 81% 91% Round2-Var81 Round1-Var94
(N409K_Q425E_S450K) 81% 84% Round2-Var82 Round1-Var94
(N27S_R64H_K393R) 47% 37% Round2-Var83 Round1-Var94
(S42A_A213N_V217L) 62% 46% Round2-Var84 Round1-Var94
(N27S_S274K_I407V) 49% 44% Round2-Var85 Round1-Var94
(I411V_Q425E_S456L) 75% 81% Round2-Var86 Round1-Var94
(A239V_K316R_E449D) 83% 72% Round2-Var87 Round1-Var94
(S147G_A239V_P348G) 18% 7% Round2-Var88 Round1-Var94
(V20L_S274G_S450K) 71% 68% Round2-Var89 Round1-Var94
(F314S_V394I_S447A) 88% 123% Round2-Var90 Round1-Var94
(R64H_E266Q_I295M) 45% 47% Round2-Var91 Round1-Var94
(N138K_I295V_I407V) 50% 51% Round2-Var92 Round1-Var94
(I15L_P348G_Q432E) 18% 13% *Mutations are noted as follows:
reference gene-original amino acid-position-new amino acid: For
example the mutation of an alanine at position 33 to a glycine for
variant 94 from the first round of directed evolution of UGT76G1 is
noted as Round1-Var94 (A33G)
[0612] Modeling of these results allowed to obtain a ranking of the
effect of each mutation. The following mutations were determined as
being beneficial for activity: S42A, F46I, I190L, S274G, I295M,
K303G, F314S, K316R, K393R, V394I, 1407V, N409K, N409R, Q425E,
Q432E, S447A, S456L.
Example 42
[0613] In vivo production of AtSUS
[0614] AtSUS
[0615] >gi|79328294|ref|NP_001031915.1| sucrose synthase 1
[Arabidopsis thaliana]
TABLE-US-00066 MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQII
AEFEALPEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYL
RVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPT
LHKYIGNGVDFLNRHLSAKLFHDKESLLPLLKFLRLHSHQGKNLMLSEKI
QNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAERVL
DMIRLLLDLLEAPDPCTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPD
TGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRUPDAVGTICGE
RLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELS
KELNGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIY
WKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTA
FTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIE
ELLYSDVENKEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLR
ELANLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDR
VRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPAE
IIVHGKSGFHIDPYHGDQAADTLADFFTKCKEDPSHWDEISKGGLQRIEE
KYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEARRYLEMFYALKYRPLAQA VPLAQDD
[0616] The synthetic gene of AtSuS that was codon optimized for
expression in E. coli and subcloned in the pET30a+ plasmid using
the NdeI and XhoI restriction sites. The pET30A+ vector containing
the AtSUS gene was used to transform electrocompetent E. coli
B121(DE3) cells. The obtained cells were grown in petri-dishes in
the presence of Kanamycin and suitable colonies were selected and
allowed to grow in liquid LB medium (erlenmeyer flasks). Glycerol
was added to the suspension as cryoprotectant and 400 .mu.L,
aliquots were stored at -20.degree. C. and at -80.degree. C.
[0617] The storage aliquots of E. coli BL21(DE3) containing the
pET30A-LAtSUS plasmids were thawed and added to 30 mL of LBGKP
medium (20 g/L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50
mM Phosphate buffer pH 7.00; 2.5 g/L glucose and 50 mg/L of
Kanamycine). This culture was allowed to shake at 135 rpm at
30.degree. C. for 8 h.
[0618] The production medium contained 60 g/L of overnight express
instant TB medium (Novagen), 10 g/L of glycerol and 50 mg/L of
Kanamycine. The preculture was added to 800 mL of this medium and
the solution was allowed to stir at 20.degree. C. while taking
samples to measure the OD and pH. The culture gave significant
growth and a good OD was obtained. After 40 h, the cells were
harvested by centrifugation and frozen to obtain 30.1 g of cell wet
weight.
[0619] Lysis was performed by Fastprep (MP Biomedicals, Lysing
matrix B, speed 6.0, 3.times.40 sec) with a cell suspension of 200
mg of cells in 1.0 mL of 50 mM Tris buffer pH 7.5. The lysate was
recovered by centrifugation and used fresh.
Example 43
[0620] Conversion of Rebaudioside A to Rebaudioside X with in situ
prepared UDP-Glucose using UGTSL2, UGT76G1-R1-F12 and AtSUS
[0621] The reaction was performed at 1 mL scale using 100 mM of
sucrose, 3 mM of MgCl.sub.2, 0.25 mM of UDP and 0.5 mM of
Rebaudioside A in potassium phosphate buffer (50 mM final
concentration, pH 7.5). The reaction was started by adding 15 .mu.L
of UGTSL2 (see EXAMPLE 27) lysate (2 U/mL), 150 .mu.L of
UGT76G1var94 (see EXAMPLE 26) (2.5 U/mL) and 15 .mu.L of AtSUS (see
EXAMPLE 42) (400 U/mL). The reaction was followed by HPLC after
quenching 125 .mu.L samples with 10 .mu.L of 2 N H.sub.2SO.sub.4
and 115 .mu.L of 60% methanol. 68% of Rebaudioside X and 26% of
Rebaudioside M2 was obtained after 21 h of reaction time, as shown
in FIG. 66.
Example 44
[0622] Directed evolution of UGT76G1 for the conversion of
Rebaudioside D to Rebaudioside X (Round 3)
[0623] The most active clone from the second round of directed
evolution of UGT76G1 (see EXAMPLE 41 round2_UGT76G1var66 containing
mutations: S42A_F46I_I407V) was chosen as baseline clone for round
3. A list of 56 mutations was established containing different
identified positive mutations from the second round and 30 new
mutations obtained by DNA2.0 ProteinGPStm strategy. This list of
mutations was subsequently used to design 92 variant genes that
contained each 3 or 4 different mutations. After codon-optimized
for expression in E. coli the genes were synthesized, subcloned in
the pET30a+ plasmid and used for transformation of E. coli BL21
(DE3) chemically competent cells. The obtained cells were grown in
Petri-dishes on solid LB medium in the presence of Kanamycin.
Suitable colonies were selected and allowed to grow in liquid LB
medium in tubes. Glycerol was added to the suspension as
cryoprotectant and 400 .mu.L aliquots were stored at -20.degree. C.
and at -80.degree. C.
[0624] These storage aliquots of E. coli BL21(DE3) containing the
pET30a+_UGT76G1var plasmids were thawed and added to LBGKP medium
(20 g/L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM
Phosphate buffer pH 7.00; 2.5 g/L glucose and 50 mg/L of
Kanamycine). This culture was allowed to shake in a 96 microtiter
plate at 30.degree. C. for 8 h.
[0625] 3.95 mL of production medium containing 60 g/L of Overnight
Express.TM. Instant TB medium (Novagen.RTM.), 10 g/L of glycerol
and 50 mg/L of Kanamycin was inoculated with 50 .mu.L of above
described culture. In a 48 deepwell plate the resulting culture was
allowed to stir at 20.degree. C. The cultures gave significant
growth and a good OD (600 nm) was obtained. After 44 h, the cells
were harvested by centrifugation and frozen.
[0626] Lysis was performed by addition of Bugbuster.RTM. Master mix
(Novagen.RTM.) to the thawed cells and the lysate was recovered by
centrifugation. Activity tests were performed with 100 .mu.L of
fresh lysate that was added to a solution of Rebaudioside D (final
concentration 0.5 mM), MgCl.sub.2 (final concentration 3 mM) and
UDP-Glucose (final concentration 2.5 mM) in 50 mM phosphate buffer
pH 7.2.
[0627] The reaction was allowed to run at 30.degree. C. and samples
were taken after 1, 2, 4, 6 and 22 h. to determine conversion and
initial rate by HPLC (CAD detection) using the analytical method
that was described above for the transformation of Rebaudioside D
to Rebaudioside X. In parallel the experiments were performed with
baseline clone, Round2-Var66. The conversion after 22 h. and
initial rate for this baseline clone was defined as 100% and the
normalized conversions and initial rates for the round 3 clones are
depicted in the following table:
TABLE-US-00067 Normalized conversion Normalized initial Clone
Mutations* Reb D to Reb X after 22 h. rate (0-4 h) Round2-Var66
UGT76G1 100% 100% (S42A_F46I_Q266E_P272A_R334K_G348P_L379G_I407V)
Baseline clone Round3-Var1 Round2-Var66 (I46F_L121I_E229A_K393R)
42% 96% Round3-Var2 Round2-Var66 (F18V_A213N_E266S) 7% 36%
Round3-Var3 Round2-Var66 (F41L_I190L_A239V_K316R) 20% 64%
Round3-Var4 Round2-Var66 (N138K_S274G_Q425E_S456L) 92% 104%
Round3-Var5 Round2-Var66 (F22Y_E229S_V407I_N409R) 15% 66%
Round3-Var6 Round2-Var66 (F150A_G216A_T355S_S447A) 15% 50%
Round3-Var7 Round2-Var66 (V394I_N409R_Q425E_S447A) 72% 97%
Round3-Var8 Round2-Var66 (Y37H_F41L_N409R_Q425E) 6% 37% Round3-Var9
Round2-Var66 (L121V_F182L_K303G_E331G) 75% 95% Round3-Var10
Round2-Var66 (S274G_K303G_N409R_Q432E) 99% 106% Round3-Var11
Round2-Var66 (F41L_K303G_F314S) 26% 67% Round3-Var12 Round2-Var66
(F22Y_R141S_T284V) 3% 15% Round3-Var13 Round2-Var66
(I190L_E229A_T284V) 31% 70% Round3-Var14 Round2-Var66
(K303G_Q425E_S447A) 109% 114% Round3-Var15 Round2-Var66
(K316R_L383V_V394I) 107% 117% Round3-Var16 Round2-Var66
(I190L_K303G_S447A_S456L) 112% 110% Round3-Var17 Round2-Var66
(N138G_V264C_A352G_S447A) 102% 107% Round3-Var18 Round2-Var66
(S274K_V407I_Q425E) 91% 107% Round3-Var19 Round2-Var66
(I190L_S274G_K393R_V394I) 120% 108% Round3-Var20 Round2-Var66
(A213N_L277I_Q425E_E449D) 79% 101% Round3-Var21 Round2-Var66
(I46L_K303G_K393R) 147% 117% Round3-Var22 Round2-Var66
(S221T_S274G_S375Q) 19% 65% Round3-Var23 Round2-Var66
(Y37H_L383V_S456L) 67% 99% Round3-Var24 Round2-Var66
(N138G_I190L_I295T_N409R) 45% 84% Round3-Var25 Round2-Var66
(A42S_S119A_K303G_V407I) 92% 99% Round3-Var26 Round2-Var66
(F22Y_I46F_I190L_V394I) 76% 95% Round3-Var27 Round2-Var66
(N138K_A213N_F314S) 83% 92% Round3-Var28 Round2-Var66
(D301N_F314S_V394I_N409R) 76% 86% Round3-Var29 Round2-Var66
(G216A_E266S_Q432E) 70% 88% Round3-Var30 Round2-Var66
(N138K_A239V_P382R_K393R) 42% 76% Round3-Var31 Round2-Var66
(I46L_S274G_K316R_S456L) 149% 109% Round3-Var32 Round2-Var66
(F18V_I190L_S375Q_S456L) 1% 2% Round3-Var33 Round2-Var66
(N138K_R141S_S274G) 18% 57% Round3-Var34 Round2-Var66
(N138K_K393R_N409R_S447A) 59% 82% Round3-Var35 Round2-Var66
(I295T_K303G_P382R_V394I) 31% 70% Round3-Var36 Round2-Var66
(N138K_I218V_S456L) 54% 81% Round3-Var37 Round2-Var66
(M145R_S274K_L383V) 1% 1% Round3-Var38 Round2-Var66
(F182L_A352G_V394I) 86% 96% Round3-Var39 Round2-Var66
(A42S_N138G_E229A_S456L) 21% 77% Round3-Var40 Round2-Var66
(R141S_I190L_E331G_Q425E) 6% 35% Round3-Var41 Round2-Var66
(E229S_K316R_T355S) 32% 81% Round3-Var42 Round2-Var66
(I46F_N138K_F292L_N409R) 30% 83% Round3-Var43 Round2-Var66
(A42S_F182L_L277I_T355S) 40% 89% Round3-Var44 Round2-Var66
(S274G_T284V_Q425E) 85% 93% Round3-Var45 Round2-Var66
(A272P_E331G_V394I_S447A) 88% 96% Round3-Var46 Round2-Var66
(S274G_F314S_Q432E_S447A) 112% 104% Round3-Var47 Round2-Var66
(L121I_K316R_S375Q_N409R) 24% 76% Round3-Var48 Round2-Var66
(L121I_N138K_F150A_K303G) 40% 83% Round3-Var49 Round2-Var66
(I46F_V264C_Q432E) 61% 98% Round3-Var50 Round2-Var66
(F150A_A272P_D301N_K316R) 44% 88% Round3-Var51 Round2-Var66
(I46L_R64V_A239V) 28% 71% Round3-Var52 Round2-Var66
(L121I_I218V_F314S) 87% 94% Round3-Var53 Round2-Var66
(I190L_G216A_E449D) 49% 90% Round3-Var54 Round2-Var66
(S274G_I295M_F314S) 128% 106% Round3-Var55 Round2-Var66
(F22Y_S274G_P382R_Q432E) 39% 48% Round3-Var56 Round2-Var66
(N138K_I190L_K334R) 93% 97% Round3-Var57 Round2-Var66
(N138G_I295M_K303G) 110% 100% Round3-Var58 Round2-Var66
(L121V_G216A_Q425E_S456L) 28% 52% Round3-Var59 Round2-Var66
(F182L_F314S_K393R) 92% 97% Round3-Var60 Round2-Var66
(R64V_K316R_N409K) 16% 54% Round3-Var61 Round2-Var66
(V264C_S274G_K393R) 102% 98% Round3-Var62 Round2-Var66
(F41L_K393R_S456L) 12% 49% Round3-Var63 Round2-Var66
(A42S_S274G_F292L_V394I) 75% 87% Round3-Var64 Round2-Var66
(I190L_S221T_E266S_S447A) 34% 71% Round3-Var65 Round2-Var66
(R64V_E229S_S274K) 12% 49% Round3-Var66 Round2-Var66
(S221T_K334R_K393R_V394I) 72% 90% Round3-Var67 Round2-Var66
(I190L_K393R_Q425E_Q432E) 101% 102% Round3-Var68 Round2-Var66
(F18V_N138K_M145R) 1% 1% Round3-Var69 Round2-Var66
(I218V_F292L_K316R_S447A) 40% 79% Round3-Var70 Round2-Var66
(L121V_E229A_K316R_Q432E) 19% 63% Round3-Var71 Round2-Var66
(Y37H_L121V_D301N) 35% 68% Round3-Var72 Round2-Var66
(N138K_V394I_Q432E_S456L) 66% 89% Round3-Var73 Round2-Var66
(T284V_I295M_A352G_L383V) 69% 89% Round3-Var74 Round2-Var66
(S119A_F150A_V394I_Q425E) 66% 88% Round3-Var75 Round2-Var66
(F18V_A239V_S447A) 8% 27% Round3-Var76 Round2-Var66
(K303G_N409R_Q432E) 84% 97% Round3-Var77 Round2-Var66
(Y37H_A272P_K334R_E449D) 75% 89% Round3-Var78 Round2-Var66
(K303G_F314S_V394I_Q425E) 121% 104% Round3-Var79 Round2-Var66
(R141S_I295T_F314S_Q432E) 9% 29% Round3-Var80 Round2-Var66
(N138K_I190L_F314S_N409R) 90% 97% Round3-Var81 Round2-Var66
(S119A_E331G_S456L) 87% 97% Round3-Var82 Round2-Var66
(K303G_F314S_K393R_S456L) 100% 100% Round3-Var83 Round2-Var66
(N138K_A352G_V407I_Q432E) 72% 95% Round3-Var84 Round2-Var66
(S274G_L277I_I295T) 34% 81% Round3-Var85 Round2-Var66
(R64V_L277I_F314S_S447A) 34% 61% Round3-Var86 Round2-Var66
(S221T_N409K_Q432E) 39% 75% Round3-Var87 Round2-Var66
(N409R_S447A_S456L) 52% 86% Round3-Var88 Round2-Var66
(K393R_Q425E_Q432E) 102% 99% Round3-Var89 Round2-Var66
(I46L_F292L_S375Q_N409K) 8% 35% Round3-Var90 Round2-Var66
(M145R_K393R_N409R) 1% 1% Round3-Var91 Round2-Var66
(S119A_M145R_T355S_P382R) 0% 1% Round3-Var92 Round2-Var66
(I190L_E229S_V264C_F314S) 64% 82% *Mutations are noted as follows:
reference gene-original amino acid-position-new amino acid: For
example the mutation of an isoleucine at position 190 to a leucine
for variant 66 from the second round of directed evolution of
UGT76G1 is noted as Round2-Var66 (I190L)
[0628] Modeling of these results allowed to obtain a ranking of the
effect of each mutation. The following mutations were determined as
being beneficial for activity:
[0629] I46L, I295M, S119A, S274G, K334R, F314S, K303G, K316R,
K393R, I190L, Q425E, Q432E, N138G, V394I, F182L, V407I, A272P,
V264C, E449D, A352G.
Example 45
[0630] Directed evolution of UGTSL2 for the conversion of
Rebaudioside A to Rebaudioside D (Round 1)
[0631] Starting from native enzyme UGTSL2 (GI_460410132) a list of
60 mutations was established containing different identified
positive mutations from the first round and new mutations obtained
by DNA2.0 ProteinGPStm strategy. This list of mutations was
subsequently used to design 92 variant genes that contained each 3
different mutations. After codon-optimized for expression in E.
coli the genes were synthesized, subcloned in the pET30a+ plasmid
and used for transformation of E. coli BL21 (DE3) chemically
competent cells. The obtained cells were grown in Petri-dishes on
solid LB medium in the presence of Kanamycin. Suitable colonies
were selected and allowed to grow in liquid LB medium in tubes.
Glycerol was added to the suspension as cryoprotectant and 400
.mu.L aliquots were stored at -20.degree. C. and at -80.degree.
C.
[0632] These storage aliquots of E. coli BL21(DE3) containing the
pET30a+_UGTSL2var plasmids were thawed and added to LBGKP medium
(20 g/L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM
Phosphate buffer pH 7.00; 2.5 g/L glucose and 50 mg/L of
Kanamycine). This culture was allowed to shake in a 96 microtiter
plate at 30.degree. C. for 8 h.
[0633] 3.95 mL of production medium containing 60 g/L of Overnight
Express.TM. Instant TB medium (Novagen.RTM.), 10 g/L of glycerol
and 50 mg/L of Kanamycin was inoculated with 50 .mu.L of above
described culture. In a 48 deepwell plate the resulting culture was
allowed to stir at 20.degree. C. The cultures gave significant
growth and a good OD (600 nm) was obtained. After 44 h, the cells
were harvested by centrifugation and frozen.
[0634] Lysis was performed by addition of Bugbuster.RTM. Master mix
(Novagen.RTM.) to the thawed cells and the lysate was recovered by
centrifugation. Activity tests were performed with 100 .mu.L of
fresh lysate that was added to a solution of Rebaudioside D (final
concentration 0.5 mM), MgCl.sub.2 (final concentration 3 mM) and
UDP-Glucose (final concentration 2.5 mM) in 50 mM phosphate buffer
pH 7.2.
[0635] The reaction was allowed to run at 30.degree. C. and samples
were taken after 2, 4, 6 and 22 h. to determine the initial rate by
HPLC (CAD detection) using the analytical method that was described
above for the transformation of Rebaudioside A to Rebaudioside D.
In parallel the experiments were performed with baseline clone,
UGTSL2. The initial rate for this baseline clone was defined as
100%. As an indication of the specificity of the clones,
Rebaudioside M2 content was determined at 100% UDP-Glucose
conversion and Rebaudioside D2 content was determined at 50%
UDP-Glucose conversion. Wherein UDP glucose conversion is defined
as: ([Reb D]/[Reb A].sub.0)+([Reb D2]/[Reb A].sub.0)+2*([Reb
M2]/[Reb A].sub.0).
[0636] The normalized initial rate, Rebaudioside M2 content at 100%
UDP-glucose conversion and Rebaudioside D2 content at 50%
UDP-glucose conversion are depicted in the following table
TABLE-US-00068 Reb D2 Normalized Reb M2 content content at 50%
initial rate at 100% UDP- UDP-Glc Clone Mutations* (0-4 h) Glc
conversion conversion UGTSL2 baseline clone 100% 100% 12.5%
Round1-Var1 UGTSL2 (L276A_N278G_T329V) 220% 98% 8.5% Round1-Var2
UGTSL2 (S19I_E259G_V270L) 0% 0% Round1-Var3 UGTSL2
(I323V_S334T_V368E) 0% 0% Round1-Var4 UGTSL2 (V125I_E259G_L393V) 0%
0% Round1-Var5 UGTSL2 (Q27R_H247P_I333L) 185% 134% 15.0%
Round1-Var6 UGTSL2 (Q27R_N325S_G387E_T392A) 148% 116% 17.0%
Round1-Var7 UGTSL2 (F253Y_N325A_K365V_G371K) 0% 0% Round1-Var8
UGTSL2 (T245R_N325A_G331A_S334T) 8% 17% Round1-Var9 UGTSL2
(G331A_N339S_G371K) 2% 3% Round1-Var10 UGTSL2 (R6H_F272L_I323V) 3%
6% Round1-Var11 UGTSL2 (R6H_F21L_T329I) 0% 0% Round1-Var12 UGTSL2
(F21L_N280P_I282L) 0% 0% Round1-Var13 UGTSL2 (T245R_V254L_I333V) 0%
1% Round1-Var14 UGTSL2 (L276A_I351L_M354L_I389L) 2% 2% Round1-Var15
UGTSL2 (S19I_I240L_I351M) 4% 9% Round1-Var16 UGTSL2
(I131V_I333V_S334T) 3% 8% Round1-Var17 UGTSL2
(S200F_A285V_I351M_P361G) 0% 0% Round1-Var18 UGTSL2
(R6H_L37F_A285L) 8% 21% Round1-Var19 UGTSL2 (H247P_N249G_K289P) 8%
17% Round1-Var20 UGTSL2 (R6H_S19I_N325A) 50% 59% Round1-Var21
UGTSL2 (N280P_K289P_T329I_V368E) 0% 0% Round1-Var22 UGTSL2
(I240L_N325S_V368E) 26% 43% Round1-Var23 UGTSL2 (A205P_T245R_K365V)
0% 0% Round1-Var24 UGTSL2 (L276A_A341V_T392A) 255% 115% 7.5%
Round1-Var25 UGTSL2 (L37F_I351L_K365V) 7% 17% Round1-Var26 UGTSL2
(T199S_E259G_T329I) 80% 90% 12.0% Round1-Var27 UGTSL2
(T245R_S258T_L405V) 7% 18% Round1-Var28 UGTSL2 (K289S_I352V_P361G)
9% 15% Round1-Var29 UGTSL2 (L37F_V254L_V270L_I323V) 0% 0%
Round1-Var30 UGTSL2 (I240L_S258T_G387E) 127% 107% 11.0%
Round1-Var31 UGTSL2 (V270I_I282L_T329V_N339S) 0% 0% Round1-Var32
UGTSL2 (H247P_T329I_I351L) 0% 3% Round1-Var33 UGTSL2
(N280P_A285L_I352V_G387E) 37% 62% Round1-Var34 UGTSL2
(S19I_I323V_N325S_P361G) 0% 0% Round1-Var35 UGTSL2
(L37F_Q65P_F272L) 14% 24% Round1-Var36 UGTSL2 (H247P_N280R_A285V)
32% 54% Round1-Var37 UGTSL2 (I240L_N339S_I352V_L405V) 0% 0%
Round1-Var38 UGTSL2 (V125I_N280P_G371K) 2% 5% Round1-Var39 UGTSL2
(F253Y_I282L_A285V) 25% 45% Round1-Var40 UGTSL2 (I282L_R312L_N325S)
4% 8% Round1-Var41 UGTSL2 (T199S_S258T_N278G) 0% 9% Round1-Var42
UGTSL2 (I114V_I351M_G387E) 0% 0% Round1-Var43 UGTSL2
(S255C_S258T_V270L) 29% 59% Round1-Var44 UGTSL2 (Q27R_R312L_T329V)
86% 92% 12.0% Round1-Var45 UGTSL2 (V254L_N339S_I345L) 0% 0% 11.0%
Round1-Var46 UGTSL2 (I333V_A341V_M354L) 84% 86% Round1-Var47 UGTSL2
(F253Y_F272L_T392A) 125% 116% 12.0% Round1-Var48 UGTSL2
(F253Y_A285L_N339S) 50% 70% Round1-Var49 UGTSL2 (K289S_I345L_G387E)
0% 2% Round1-Var50 UGTSL2 (I131V_E259G_V270I) 0% 0% Round1-Var51
UGTSL2 (F272L_N280R_T329V) 0% 4% Round1-Var52 UGTSL2
(N278G_R312L_T329I_I333L) 100% 100% 13.0% Round1-Var53 UGTSL2
(I114V_I131V_N325S) 10% 20% Round1-Var54 UGTSL2
(A205P_K289P_I333V_G371K) 0% 0% Round1-Var55 UGTSL2
(S19I_F21L_S200F) 0% 0% Round1-Var56 UGTSL2
(I131V_H247P_N278G_A285L) 109% 120% 13.0% Round1-Var57 UGTSL2
(R312L_A341V_M367V) 14% 25% Round1-Var58 UGTSL2 (N280R_I333L_M354L)
0% 1% Round1-Var59 UGTSL2 (S258T_E259G_A285V_I333V) 0% 0%
Round1-Var60 UGTSL2 (P361G_I389L_L405V) 0% 0% Round1-Var61 UGTSL2
(S255C_N280R_I345L_V368E) 0% 0% Round1-Var62 UGTSL2
(F21L_Q65P_N280R_K289S) 0% 0% Round1-Var63 UGTSL2
(V270I_M367V_V368E) 20% 32% Round1-Var64 UGTSL2 (T199S_V254L_A285L)
0% 0% Round1-Var65 UGTSL2 (S255C_N280P_G331A) 73% 82% 11.5%
Round1-Var66 UGTSL2 (N249G_K365V_M367V_I389L) 0% 0% Round1-Var67
UGTSL2 (S200F_I333L_I351L) 0% 0% Round1-Var68 UGTSL2
(N249G_V270L_K289S) 13% 24% Round1-Var69 UGTSL2 (I114V_V125I_N249G)
6% 9% Round1-Var70 UGTSL2 (V125I_K289P_N325A) 0% 1% Round1-Var71
UGTSL2 (N249G_N325A_I352V) 43% 76% 11.5% Round1-Var72 UGTSL2
(V270I_A285V_M354L) 196% 158% 11.5% Round1-Var73 UGTSL2
(Q65P_V254L_M367V) 0% 0% Round1-Var74 UGTSL2 (V270I_K289P_S334T) 0%
0% Round1-Var75 UGTSL2 (T199S_A205P_L393V) 0% 0% Round1-Var76
UGTSL2 (V125I_I345L_M367V_T392A) 8% 19% Round1-Var77 UGTSL2
(A205P_I323V_T392A) 0% 0% Round1-Var78 UGTSL2 (F21L_L37F_I131V) 0%
0% Round1-Var79 UGTSL2 (F272L_I282L_A341V_I351L) 0% 2% Round1-Var80
UGTSL2 (N278G_I352V_I389L) 95% 113% 11.5% Round1-Var81 UGTSL2
(I114V_G331A_A341V_L405V) 8% 20% Round1-Var82 UGTSL2
(Q27R_Q65P_I351M) 0% 0% Round1-Var83 UGTSL2 (R6H_T329V_M354L_L393V)
77% 100% 10.5% Round1-Var84 UGTSL2 (S200F_G331A_L393V) 0% 0%
Round1-Var85 UGTSL2 (T199S_K289S_R312L_I351M) 0% 0% Round1-Var86
UGTSL2 (Q65P_A205P_L405V) 0% 0% Round1-Var87 UGTSL2
(V270L_I345L_K365V) 0% 0% Round1-Var88 UGTSL2 (S200F_F253Y_S255C)
0% 0% Round1-Var89 UGTSL2 (I114V_G371K_I389L) 0% 3% Round1-Var90
UGTSL2 (L276A_I333L_S334T_L393V) 75% 94% 11.5% Round1-Var91 UGTSL2
(I240L_S255C_P361G) 5% 13% Round1-Var92 UGTSL2 (Q27R_T245R_L276A)
51% 81% 12.0% *Mutations are noted as follows: reference
gene-original amino acid-position-new amino acid: For example the
mutation of an isoleucine at position 240 to a Leucine for UGTSL2
is noted as UGTSL2 (I240L)
[0637] Modeling of these results allowed to obtain a ranking of the
effect of each mutation. The following mutations were determined as
being beneficial for activity: L276A, T392A, Q27R, N278G, T329V,
A341V, I333L, G387E, H247P, M354L, A285V, V270I, N325S, I240L,
F253Y, A285L, 1352V.
[0638] The following mutations were determined as being beneficial
for lower Rebaudioside M2 formation:
[0639] Q27R, N325S, G387E, I333L, H247P, T329I, R312L, T199S,
E259G, S334T, I131V, A285L, I389L, L393V, V254L, N339S, I345L,
T245R.
Example 46
[0640] Conversion of Rebaudioside A to Rebaudioside I using
UGT76G1
[0641] The reaction was conducted using UGT76G1-R1-F12 (also known
as UGT76G1 var94 (see EXAMPLE 26))
[0642] The total volume of the reaction was 40 mL with the
following composition: 50 mM potassium phosphate buffer pH 7.5, 3
mM MgCl.sub.2, 2.5 mM UDP-glucose, 0.5 mM Rebaudioside A and 4 mL
of UGT76G1-R1-F12 lysate (2.5 U/mL). The reaction was run at
30.degree. C. on an orbitary shaker at 135 rpm. For sampling 125
.mu.L of the reaction mixture was quenched with 10 .mu.L of 2N
H.sub.2SO.sub.4 and 115 .mu.L of methanol/water (7/3). The samples
were immediately centrifuged and kept at 10.degree. C. before
analysis by LC-MS. An Agilent 1200 series HPLC system, equipped
with binary pump (G1312B), autosampler (G1367D), thermostatted
column compartment (G1316B), DAD detector (G1315C), connected with
Agilent 6110A MSD, and interfaced with "LC/MSD Chemstation"
software, was used.
[0643] Instrument Conditions
TABLE-US-00069 Column Phenomenex Kinetex 2.6u C18 100A, 4.6 mm
.times. 150 mm, 2.6 .mu.m Column Temperature 55.degree. C.
Detection DAD at 210 nm bw 360 nm MSD (Scan and SIM mode) Mode:
ES-API, Negative Polarity Drying gas flow: 13.0 L/min Nebulizer
pressure: 30 psig Drying gas temperature: 270.degree. C. Analysis
duration 20 min Injected volume 2 .mu.L Flow rate 0.8 mL/min
[0644] Mobile Phase Gradient Program
TABLE-US-00070 Time (min) A (%): Formic acid 0.1% B (%):
Acetonitrile 0 76 24 8.5 76 24 10.0 71 29 16.5 70 30
[0645] The reaction profile shown in FIG. 67a was obtained:
[0646] After 42 h. of reaction, 20 mL of the reaction mixture was
quenched with 20 mL of ethanol and used for structure
elucidation.
[0647] In similar manner the best clones of UGT76G1 directed
evolution round 2 (UGT76G1-R2-B9 identified above as
"Round2-Var66", see EXAMPLE 41) and round 3 (UGT76G1-R3-G3
identified above as "Round3-Var21", see EXAMPLE 44) and native
UGT76G1 (see EXAMPLE 26) were tested for the conversion of
Rebaudioside A to Rebaudioside I and the activities shown in FIG.
67b were determined.
Example 47
[0648] Isolation and Characterization of Reb I
[0649] Crude Reaction Sample.
[0650] The sample, Lot Crude CB-2977-198, used for isolation, was
prepared according to Example 46 with UGT76G1.
[0651] HPLC Analysis.
[0652] Preliminary HPLC analyses of samples were performed using a
Waters 2695 Alliance System with the following method: Phenomenex
Synergi Hydro-RP, 4.6.times.250 mm, 4 .mu.m (p/n 00G-4375-E0);
Column Temp: 55.degree. C.; Mobile Phase A: 0.0284% NH.sub.4OAc and
0.0116% HOAc in water; Mobile Phase B: Acetonitrile (MeCN); Flow
Rate: 1.0 mL/min; Injection volume: 10 .mu.L. Detection was by UV
(210 nm) and CAD
[0653] Gradient:
TABLE-US-00071 Time (min) % A % B 0.0-8.5 75 25 10.0 71 29 16.5 70
30 18.5-24.5 66 34 26.5-29.0 48 52 31-37 30 70 38 75 25
[0654] Isolation by HPLC.
[0655] The purification was performed using a Waters Atlantis dC18
(30.times.100 mm, 5 .mu.m, p/n 186001375) column with isocratic
mobile phase conditions of 80:20 water/MeCN. Flow rate was
maintained at 45 mL/min and injection load was 180 mg. Detector
wavelength was set at 210 nm.
[0656] The analyses of fractions were performed using a Waters
Atlantis dC18 (4.6.times.150 mm, 5 .mu.m, p/n 186001342) column;
Mobile Phase A: water; Mobile Phase B: MeCN; Flow Rate: 1 mL/min;
Isocratic mobile phase conditions: 75:25 AB for 30 min.
[0657] MS and MS/MS.
[0658] MS and MS/MS data were generated with a Waters QT of Micro
mass spectrometer equipped with an electrospray ionization source.
The sample was analyzed by negative ESI. The sample was diluted to
a concentration of 0.25 mg/mL with H.sub.2O:MeCN (1:1) and
introduced via flow injection for MS data acquisition. The sample
was diluted further to 0.01 mg/mL to yield good s/n to tune for
MS/MS and acquired by direct infusion. The collision energy was set
to 60 V in order to acquire MS/MS data with increased fragment ion
peaks due to the nature of the molecule
[0659] NMR.
[0660] The sample was prepared by dissolving .about.1.0 mg in 180
.mu.L of pyridine-d.sub.5+TMS, and NMR data were acquired on a
Bruker Avance 500 MHz instrument with either a 2.5 mm inverse probe
or a 5 mm broad band probe. The 13C and HMBC NMR data were acquired
at Rensselaer Polytechnic Institute using their Bruker Avance 600
MHz and 800 MHz instruments with 5 mm cryo-probe, respectively. The
.sup.1H and .sup.13C NMR spectra were referenced to the TMS
resonance (.delta..sub.H0.00 ppm and .delta..sub.C 0.0 ppm).
[0661] Isolation of Reb I was performed using a semi-synthetic
steviol glycoside mixture, Lot number CB-2977-198. The material was
analyzed by HPLC as described above. The Reb I peak was observed at
a retention time (t.sub.R) of approximately 17 min as shown in FIG.
28.
[0662] Results and Discussion
[0663] The reb I peak was isolated from the reaction crude as
described above and shown in FIG. 29. The isolated fraction was
pooled and lyophilized. Purity of the final product was 91% as
confirmed by LC-CAD using the method described above (FIG. 30).
Approximately 1 mg of Reb I was provided for spectroscopic and
spectrometric analyses.
[0664] Mass Spectrometry.
[0665] The ESI-TOF mass spectrum acquired by infusing a sample of
reb I showed a [M-H].sup.- ion at m/z 1127.4741 (FIG. 31). The mass
of the [M-H].sup.- ion was in good agreement with the molecular
formula C.sub.50H.sub.79O.sub.28 (calcd for
C.sub.50H.sub.79O.sub.28: 1127.4758, error: -1.5 ppm) expected for
reb I (FIG. 32). The MS data confirmed that reb I has a nominal
mass of 1128 Daltons with the molecular formula,
C.sub.50H.sub.80O.sub.28.
[0666] The MS/MS spectrum of reb I, selecting the [M-H].sup.- ion
at m/z 1127.4 for fragmentation, indicated loss of two sugar units
at m/z 803.5301, however did not show additional fragmentation with
collision energy of 30 V (FIG. 33). When higher collision energy
was applied (60 V) (FIG. 34), the parent ion was not observed but
sequential loss of three sugar units at m/z 641.4488, 479.3897, and
317.3023 were observed from m/z 803.5301
[0667] NMR Spectroscopy.
[0668] A series of NMR experiments including .sup.1H NMR (FIGS.
35-37), .sup.13C NMR (FIGS. 38-39), .sup.1H-.sup.1H COSY (FIG. 40),
HSQC-DEPT (FIG. 41), HMBC (FIGS. 42-43), NOESY (FIGS. 44-45), and
1D TOCSY (FIGS. 46-50) were performed to allow assignment of reb
I.
[0669] In the .sup.1H NMR spectrum of reb I acquired at 300 K (FIG.
35), one of the anomeric protons was completely obscured by the
water resonance. Therefore, .sup.1H NMR spectrum of the sample was
acquired at lower temperature (292 K), to shift out the water
resonance, and at this temperature anomeric proton was sufficiently
resolved (FIGS. 36-37). Thus, all other NMR data of reb I was
acquired at 292 K.
[0670] The 1D and 2D NMR data indicated that the central core of
the glycoside is a diterpene. An HMBC correlation from the methyl
protons at .delta..sub.H 1.22 to the carbonyl at .delta..sub.C
176.9 allowed assignment of one of the tertiary methyl groups
(C-18) as well as C-19 and provided a starting point for the
assignment of the rest of the aglycone. Additional HMBC
correlations from the methyl protons (H-18) to carbons at
.delta..sub.C 38.5, 44.0, and 57.2 allowed assignment of C-3, C-4,
and C-5. Analysis of the .sup.1H-.sup.13C HSQC-DEPT data indicated
that the carbon at .delta..sub.C 38.5 was a methylene group and the
carbon at .delta..sub.C 57.2 was a methine which were assigned as
C-3 and C-5, respectively. This left the carbon at .delta..sub.C
44.0, which did not show a correlation in the HSQC-DEPT spectrum,
to be assigned as the quaternary carbon, C-4. The .sup.1H chemical
shifts for C-3 (.delta..sub.H 1.02 and 2.35) and C-5 (.delta..sub.H
1.03) were assigned using the HSQC-DEPT data. A COSY correlation
between one of the H-3 protons (.delta..sub.H 1.02) and a proton at
.delta..sub.H 1.44 allowed assignment of one of the H-2 protons
which in turn showed a correlation with a proton at .delta..sub.H
0.74 which was assigned to H-1. The remaining .sup.1H and .sup.13C
chemical shifts for C-1 and C-2 were then assigned on the basis of
additional COSY and HSQC-DEPT correlations and are summarized in
the table below.
TABLE-US-00072 .sup.1H and .sup.13C NMR (500 and 150 MHz,
pyridine-d.sub.5), assignments of the Rebaudioside I aglycone.
Position .sup.13C .sup.1H 1 40.7 0.74 t (11.6) 1.75 m 2 19.4 1.44 m
2.20 m 3 38.5 1.02 m 2.35 m 4 44.0 -- 5 57.2 1.03 m 6 22.2 1.90 m
2.33 m 7 41.7 1.29 m 1.31 m 8 42.3 -- 9 54.1 0.88 d (6.3) 10 39.8
-- 11 20.5 1.67 m 1.70 m 12 37.3 1.98 m 2.28 m 13 86.7 -- 14 44.3
1.78 m 2.59 d (11.9) 15 47.6 2.04 brs 16 154.0 -- 17 104.8 5.02 s
5.67 s 18 28.4 1.22 s 19 176.9 -- 20 15.7 1.26 s
[0671] The other tertiary methyl singlet, observed at .delta..sub.H
1.26, showed HMBC correlations to C-1 and C-5 and was assigned as
H-20. The methyl protons showed additional HMBC correlations to a
quaternary carbon (.delta..sub.C 39.8) and a methine carbon
(.delta..sub.C 54.1) which were assigned as C-10 and C-9,
respectively. COSY correlations between H-5 (.delta..sub.H 1.03)
and protons at .delta..sub.H 1.90 and 2.33 then allowed assignment
of the H-6 protons which in turn showed correlations to protons at
.delta..sub.H 1.29 and 1.31 which were assigned to H-7. The
.sup.13C chemical shifts for C-6 (.delta..sub.C 22.2) and C-7
(.delta..sub.C 41.7) were then determined from the HSQC-DEPT data.
COSY correlations between H-9 (.delta..sub.H 0.88) and protons at
.delta..sub.H 1.67 and 1.70 allowed assignment of the H-11 protons
which in turn showed COSY correlations to protons at .delta..sub.H
1.98 and 2.28 which were assigned as the H-12 protons. The
HSQC-DEPT data was then used to assign C-11 (.delta..sub.C 20.5)
and C-12 (.delta..sub.C 37.3). The olefinic protons observed at
.delta..sub.H 5.02 and 5.67 showed HMBC correlations to a
quaternary carbon at .delta..sub.C 86.7 (C-13) and thus were
assigned to H-17 (.delta..sub.C 104.8 via HSQC-DEPT). The methine
proton H-9 showed HMBC correlations to carbons at .delta..sub.C
42.3, 44.3 and 47.6 which were assigned as C-8, C-14 and C-15,
respectively. The .sup.1H chemical shifts at C-14 (.delta..sub.H
1.78 and 2.59) and C-15 (.delta..sub.H 2.04) were assigned using
the HSQC-DEPT data. Additional HMBC correlations from H-9 to C-11
and H-12 to C-9 further confirmed the assignments made above. HMBC
correlations observed from H-14 to a quaternary carbon at
.delta..sub.C 154.0 allowed the assignment of C-16 to complete the
assignment of the central core.
[0672] Correlations observed in the NOESY spectrum were used to
assign the relative stereochemistry of the central diterpene core.
In the NOESY spectrum, NOE correlations were observed between H-14
and H-20 indicating that H-14 and H-20 are on the same face of the
rings. Similarly, NOE correlations were observed between H-9 and
H-5 as well as H-5 and H-18. NOE correlations between H-9 and H-14
were not observed. The NOESY data thus indicate that H-5, H-9 and
H-18 were on the opposite face of the rings compared to H-14 and
H-20 as presented in the figure below. These data thus indicate
that the relative stereochemistry in the central core was retained
during the glycosylation step.
[0673] Analysis of the .sup.1H-.sup.13C HSQC-DEPT data for reb I
confirmed the presence of five anomeric protons. All five anomeric
protons were resolved in the spectra acquired at 292 K at
.delta..sub.H 6.14 (.delta..sub.C 95.3), 5.57 (.delta..sub.C
104.6), 5.38 (.delta..sub.C 104.7), 5.29 (.delta..sub.C 105.0), and
5.06 (Sc 98.0). Additionally, all five anomeric protons had large
couplings (7.7 Hz-8.2 Hz) indicating that they had
n-configurations. The anomeric proton observed at .delta..sub.H
6.14 showed an HMBC correlation to C-19 which indicated that it
corresponds to the anomeric proton of Glc.sub.I. Similarly, the
anomeric proton observed at .delta..sub.H 5.06 showed an HMBC
correlation to C-13 allowing it to be assigned as the anomeric
proton of Glc.sub.II.
[0674] The Glc.sub.I anomeric proton (.delta..sub.H 6.14) showed a
COSY correlation to a proton at .delta..sub.H 4.18 which was
assigned as Glc.sub.I H-2. Due to data overlap the COSY spectrum
did not allow assignment of H-3 or H-4. Therefore, a series of 1D
TOCSY experiments were performed using selective irradiation of the
Glc.sub.I anomeric proton with several different mixing times (FIG.
46). In addition to confirming the assignment for Glc.sub.I H-2,
the TOCSY data showed protons at .delta..sub.H 4.27, 4.25, and 3.93
which were assigned as H-3, H-4 and H-5, respectively. The proton
observed at .delta..sub.H 4.37 in the TOCSY spectrum was assigned
to one of the Glc.sub.I H-6 protons. The other H-6 methylene proton
at .delta..sub.H 4.27 was assigned based on COSY correlation from
H-5 to .delta..sub.H 4.27. The .sup.13C chemical shifts for
Glc.sub.I C-2 (.delta..sub.C 72.5), C-3 (.delta..sub.C 89.4), C-4
(.delta..sub.C 69.2), C-5 (.delta..sub.C 78.2-78.8) and C-6
(.delta..sub.C 61.7) were assigned using the HSQC-DEPT data. HMBC
correlations from H-1 to C-3 and H-4 to C-6 further confirmed the
assignments made above to complete the assignment of Glc.sub.I.
[0675] Of the four remaining unassigned glucose moieties one was
assigned as a substituent at C-3 of Glc.sub.I on the basis of HMBC
correlations. The anomeric proton observed at .delta..sub.H 5.29
showed an HMBC correlation to Glc.sub.I C-3 and was assigned as the
anomeric proton of Glc.sub.V. The reciprocal HMBC correlation from
Glc.sub.I H-3 to the anomeric carbon of Glc.sub.V was also
observed.
[0676] A summary of the .sup.1H and .sup.13C chemical shifts for
the glycoside at C-19 are shown in the following table:
TABLE-US-00073 .sup.1H and .sup.13C NMR (500 and 150 MHz,
pyridine-d.sub.5) assignments of Rebaudioside I C-19 glycoside.
Position .sup.13C .sup.1H Glc.sub.I-1 95.3 6.14 d (8.2) Glc.sub.I-2
72.5 4.18 m Glc.sub.I-3 89.4 4.27 m Glc.sub.I-4 69.2 4.25 m
Glc.sub.I-5 78.2-78.8.sup..dagger. 3.93 m Glc.sub.I-6 61.7 4.27 m,
4.37 m Glc.sub.V-1 105.0 5.29 d (7.9) Glc.sub.V-2 75.3 or 75.5.sup.
4.04 m Glc.sub.V-3 78.2-78.6.sup..dagger. 4.27 m Glc.sub.V-4 71.5
or 71.6.sup. 4.12 m Glc.sub.V-5 78.5 or 78.6.sup..dagger. 4.05 m
Glc.sub.V-6 62.3 or 62.4.sup. 4.26 m, 4.56 m .sup..dagger.Five
carbon resonances in the range of 78.2-78.8 (78.16, 78.47, 78.50,
78.55, and 78.77), hence chemical shift could not be unequivocally
assigned.
[0677] A summary of key HMBC and COSY correlations used to assign
the C-19 glycoside region are provided below.
##STR00016##
[0678] The anomeric proton of Glc.sub.V (.delta..sub.H 5.29) showed
a COSY correlation with a proton at .delta..sub.H 4.04 which was
assigned as Glc.sub.V H-2. Glc.sub.V C-2 (.delta..sub.C 75.3 or
75.5) was then assigned using the HSQC-DEPT data. Due to overlap in
the data the COSY spectrum did not allow assignment of the
remaining protons. Therefore, a series of 1D TOCSY experiments were
performed using selective irradiation of the Glc.sub.V anomeric
proton with several different mixing times (FIG. 47). In addition
to confirming the assignments for Glc.sub.V H-2, the TOCSY data
allowed assignment of Glc.sub.V H-3 (.delta..sub.H 4.27), H-4
(.delta..sub.H 4.12), and H-5 (.delta..sub.H 4.05). The proton
observed at .delta..sub.H 4.56 in the TOCSY spectrum was assigned
to one of the Glc.sub.V H-6 protons. The other H-6 methylene proton
at .delta..sub.H 4.26 was assigned based on COSY correlation from
H-5 to .delta..sub.H 4.26. The .sup.13C chemical shifts for
Glc.sub.V C-3 (.delta..sub.C 78.2-78.6), C-4 (.delta..sub.C 71.5 or
71.6), C-5 (.delta..sub.C 78.5 or 78.6) and C-6 (.delta..sub.C 62.3
or 62.4) were assigned using the HSQC-DEPT data to complete the
assignment of Glc.sub.V.
[0679] Assignment of Glc.sub.II was carried out in a similar
manner. The Glc.sub.II anomeric proton (.delta..sub.H 5.06) showed
a COSY correlation to a proton at .delta..sub.H 4.34 which was
assigned as Glc.sub.II H-2 and in turn showed a COSY correlation to
a proton at .delta..sub.H 4.20 (Glc.sub.II H-3) which showed an
additional correlation with a proton at .delta..sub.H 3.97
(Glc.sub.II H-4) which also showed a COSY correlation to a proton
at .delta..sub.H 3.80 (Glc.sub.II H-5). H-5 showed additional COSY
correlations to protons at .delta..sub.H 4.18 and 4.49 which were
assigned to H-6. A series of 1D TOCSY experiments were also
performed using selective irradiation of the Glc.sub.II anomeric
proton with several different mixing times (FIG. 48). The TOCSY
data confirmed the above proton assignments. Assignment of the
.sup.13C chemical shifts for Glc.sub.II C-2 (.delta..sub.C 80.2),
C-3 (.delta..sub.C 87.5), C-4 (.delta..sub.C 70.1), C-5
(.delta..sub.C 77.6) and C-6 (.delta..sub.C 62.5) was based on
HSQC-DEPT data. HMBC correlations from Glc.sub.II H-3 to C-2 and
C-4 and also from Glc.sub.II H-4 to C-3, C-5 and C-6 confirmed the
assignments made above to complete the assignment of
Glc.sub.II.
[0680] The remaining two unassigned glucose moieties were assigned
as substituents at C-2 and C-3 of Glc.sub.II on the basis of HMBC
correlations. The anomeric proton observed at .delta..sub.H 5.57
showed an HMBC correlation to Glc.sub.II C-2 and was assigned as
the anomeric proton of Glc.sub.III. The anomeric proton observed at
.delta..sub.H 5.38 showed an HMBC correlation to Glc.sub.II C-3 and
was assigned as the anomeric proton of Glc.sub.IV. The reciprocal
HMBC correlations from Glc.sub.II H-2 to the anomeric carbon of
Glc.sub.III and from Glc.sub.II H-3 to the anomeric carbon of
Glc.sub.IV were also observed.
[0681] The anomeric proton of Glc.sub.III (.delta..sub.H 5.57)
showed a COSY correlation with a proton at .delta..sub.H 4.21 which
was assigned as Glc.sub.III H-2. Glc.sub.III C-2 (.delta..sub.C
76.3) was then assigned using the HSQC-DEPT data. Due to data
overlap the COSY spectrum did not allow assignment of the remaining
protons. Therefore, a series of 1D TOCSY experiments were performed
using selective irradiation of the Glc.sub.III anomeric proton with
several different mixing times (FIG. 49). In addition to confirming
the assignments for Glc.sub.III H-2, the TOCSY data allowed
assignment of Glc.sub.III H-3 (.delta..sub.H4.27), H-4
(.delta..sub.H 4.25) and H-5 (.delta..sub.H 3.94). The protons
observed at .delta..sub.H 4.41 and .delta..sub.H 4.53 in the TOCSY
spectrum were assigned as the Glc.sub.III H-6 protons. The .sup.13C
chemical shifts for C-3 (.delta..sub.C 78.2-78.6), C-4
(.delta..sub.C 72.1), C-5 (.delta..sub.C 78.2-78.8) and C-6
(.delta..sub.C 63.1) were assigned using the HSQC-DEPT data. HMBC
correlations from H-5 to a carbon at .delta..sub.C 63.1 further
confirmed the assignment of Glc.sub.III C-6 to complete the
assignment of Glc.sub.II'.
[0682] The anomeric proton of Glc.sub.IV (.delta..sub.H 5.38)
showed a COSY correlation with a proton at .delta..sub.H 4.01 which
was assigned as Glc.sub.II/H-2. Glc.sub.II, C-2 (.delta..sub.C 75.3
or 75.5) was then assigned using the HSQC-DEPT data. Due to data
overlap the COSY spectrum did not allow assignment of the remaining
protons. Therefore a series of 1D TOCSY experiments were performed
using selective irradiation of the Glc.sub.IV anomeric proton with
several different mixing times (FIG. 50). In addition to confirming
the assignments for Glc.sub.IV H-2, the 1D TOCSY data allowed
assignment of H-3 (.delta..sub.H 4.28), H-4 (.delta..sub.H 4.11),
H-5 (.delta..sub.H 4.13) and H-6 (.delta..sub.H 4.25 and 4.58). The
proton at .delta..sub.H 4.25 also showed COSY correlation with
.delta..sub.H 4.58 further confirmed that these protons belong to
H-6. The .sup.13C chemical shifts for C-3 (.delta..sub.C
78.2-78.6), C-4 (.delta..sub.C 72.1), C-5 (.delta..sub.C 78.2-78.6)
and C-6 (.delta..sub.C 62.3 or 62.4) were assigned using the
HSQC-DEPT data. HMBC correlations from H-4 to C-6 and H-5 to C-1
further confirmed the assignment of Glc.sub.II, C-6 to complete the
assignment of Glc.sub.IV.
[0683] A summary of the .sup.1H and .sup.13C chemical shifts for
the glycoside at C-13 are found are shown below:
TABLE-US-00074 .sup.1H and .sup.13C NMR (500 and 150 MHz,
pyridine-d.sub.5) assignments of the Rebaudioside I C-13 glycoside.
Position .sup.13C .sup.1H Glc.sub.II-1 98.0 5.06 d (7.9)
Glc.sub.II-2 80.6 4.34 m Glc.sub.II-3 87.5 4.20 m Glc.sub.II-4 70.1
3.97 m Glc.sub.II-5 77.6 3.80 m Glc.sub.II-6 62.5 4.18 m, 4.49 m
Glc.sub.III-1 104.6 5.57 d (7.7) Glc.sub.III-2 76.3 4.21 m
Glc.sub.III-3 78.2-78.6.sup..dagger. 4.27 m Glc.sub.III-4 72.1 4.25
m Glc.sub.III-5 78.2-78.8.sup..dagger. 3.94 m Glc.sub.III-6 63.1
4.41 m, 4.53 m Glc.sub.IV-1 104.7 5.38 d (7.9) Glc.sub.IV-2 75.3 or
75.5 4.01 m Glc.sub.IV-3 78.2-78.6.sup..dagger. 4.28 m Glc.sub.IV-4
72.1 4.11 m Glc.sub.IV-5 78.2-78.6.sup..dagger. 4.13 m Glc.sub.IV-6
62.3 or 62.4 4.25 m, 4.58 m .sup..dagger.Five carbon resonances in
the range of 78.2-78.8 (78.16, 78.47, 78.50, 78.55, and 78.77),
hence chemical shift could not be unequivocally assigned.
[0684] A summary of key HMBC and COSY correlations used to assign
the C-13 glycoside region are provided below.
##STR00017##
[0685] NMR and MS analyses of rebaudioside I, reb I, allowed the
full assignment of structure, shown below. The name of the chemical
compound is
(13-[(2-O-.beta.-D-glucopyranosyl-3-O-.beta.-D-glucopyranosyl)-.beta.--
D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic
acid-(3-O-.beta.-D-glucopyranosyl)-.beta.-D-glucopyranosyl)
ester].
##STR00018##
Example 48
[0686] Directed evolution of UGTSL2 for the conversion of
Rebaudioside A to Rebaudioside D (Round 2)
[0687] Taking the native enzyme UGTSL2 (GI 460410132) as baseline,
a list of 23 mutations was established containing different
identified positive mutations for activity from the first round
(EXAMPLE 45) and new mutations obtained by DNA2.0 ProteinGPS.TM.
strategy. This list of mutations was subsequently used to design 46
variant genes that contained each 3 different mutations. After
codon-optimized for expression in E. coli the genes were
synthesized, subcloned in the pET30a+ plasmid and used for
transformation of E. coli BL21 (DE3) chemically competent cells.
The obtained cells were grown in Petri-dishes on solid LB medium in
the presence of Kanamycin. Suitable colonies were selected and
allowed to grow in liquid LB medium in tubes. Glycerol was added to
the suspension as cryoprotectant and 400 .mu.L aliquots were stored
at -20.degree. C. and at -80.degree. C.
[0688] These storage aliquots of E. coli BL21(DE3) containing the
pET30a+_UGTSL2var plasmids were thawed and added to LBGKP medium
(20 g/L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM
Phosphate buffer pH 7.00; 2.5 g/L glucose and 50 mg/L of
Kanamycine). This culture was allowed to shake in a 96 microtiter
plate at 30.degree. C. for 8 h.
[0689] 3.95 mL of production medium containing 60 g/L of Overnight
Express.TM. Instant TB medium (Novagen.RTM.), 10 g/L of glycerol
and 50 mg/L of Kanamycin was inoculated with 50 .mu.L of above
described culture. In a 48 deepwell plate the resulting culture was
allowed to stir at 20.degree. C. The cultures gave significant
growth and a good OD (600 nm) was obtained. After 44 h, the cells
were harvested by centrifugation and frozen.
[0690] Lysis was performed by addition of Bugbuster.RTM. Master mix
(Novagen.RTM.) to the thawed cells and the lysates were recovered
by centrifugation.
[0691] In order to measure the activity of the variants for the
transformation of Rebaudioside A to Rebaudioside D, 100 .mu.L of
fresh lysate was added to a solution of Rebaudioside A (final
concentration 0.5 mM), MgCl.sub.2 (final concentration 3 mM) and
UDP-Glucose (final concentration 2.5 mM) in 50 mM phosphate buffer
pH 7.2. The reaction was allowed to run at 30.degree. C. and
samples were taken after 2, 4, 6 and 22 h. to determine the initial
rates after HPLC analysis (CAD detection) using the analytical
method that was described above for the transformation of
Rebaudioside A to Rebaudioside D.
[0692] In parallel for the most active clones, 100 .mu.L of fresh
lysate was added to a solution of Rebaudioside D (final
concentration 0.5 mM), MgCl.sub.2 (final concentration 3 mM) and
UDP-Glucose (final concentration 2.5 mM) in 50 mM phosphate buffer
pH 7.2. The reaction was allowed to run at 30.degree. C. and
samples were taken after 2, 4, 6 and 22 h. to determine the initial
rates for Rebaudioside D conversion after HPLC analysis (CAD
detection).
[0693] Apart from the new variants, both experiments were also
performed with baseline clone, UGTSL2. The initial rates for the
conversion of Rebaudioside A or Rebaudioside D for this baseline
clone were defined as 100%.
[0694] Activity of each clone was defined as normalized activity
compared to baseline clone UGTSL2 whereas specificity of each clone
was expressed as the ratio between the initial rates for the
conversion of Rebaudioside A and Rebaudioside D.
[0695] The normalized initial rate for the conversion of
Rebaudioside A and the ratio between the initial rates for the
conversion of Rebaudioside A and Rebaudioside D are depicted in the
following table.
TABLE-US-00075 Ratio between initial Normalized initial rates for
the conversion rate for conversion of of Rebaudioside A and Clone
Mutations* Rebaudioside A Rebaudioside D UGTSL2 Baseline clone 100%
1.67 Round2-var1 UGTSL2 (Q27R_V270I_A285V) 188% 1.21 Round2-var2
UGTSL2 (F253Y_S255C_M354L) 5% Nd Round2-var3
UGTSL2_S255C_I352V_L393V 28% Nd Round2-var4 UGTSL2_R6H_N278G_T329I
7% Nd Round2-var5 UGTSL2_H247P_V270I_A285L 75% 1.27 Round2-var6
UGTSL2_I240L_T392A_L393V 114% 1.85 Round2-var7
UGTSL2_A285L_R312L_T392A 135% 1.66 Round2-var8
UGTSL2_Q27R_G387E_T392A 164% 1.65 Round2-var9
UGTSL2_Q27R_N278G_A341V 178% 3.13 Round2-var10
UGTSL2_I240L_A285L_N325S 9% Nd Round2-var11
UGTSL2_S255C_S258T_N325S 26% Nd Round2-var12
UGTSL2_Q27R_N325S_I352V 6% Nd Round2-var13 UGTSL2_N325S_A341V_M354L
116% 1.89 Round2-var14 UGTSL2_S255C_A285V_T392A 98% 2.63
Round2-var15 UGTSL2_A285L_A341V_I352V 26% Nd Round2-var16
UGTSL2_F253Y_G387E_L393V 88% 1.69 Round2-var17
UGTSL2_V270I_T329I_L393V 88% 2.16 Round2-var18
UGTSL2_H247P_I333L_L393V 197% 1.75 Round2-var19
UGTSL2_L276A_R312L_N325S 53% 1.72 Round2-var20
UGTSL2_V270I_T329V_M354L 30% Nd Round2-var21
UGTSL2_A285V_I352V_G387E 30% Nd Round2-var22
UGTSL2_I240L_H247P_L276A 76% 2.00 Round2-var23
UGTSL2_A285V_R312L_T329I 4% Nd Round2-var24
UGTSL2_I240L_M354L_G387E 8% Nd Round2-var25
UGTSL2_N278G_R312L_I333L 50% 1.57 Round2-var26
UGTSL2_L276A_T329I_I352V 0% Nd Round2-var27
UGTSL2_L276A_T329V_G387E 73% Nd Round2-var28 UGTSL2_R6H_Q27R_L393V
9% Nd Round2-var29 UGTSL2_H247P_S258T_T329I 129% 1.21 Round2-var30
UGTSL2_N278G_N325S_T392A 206% 2.06 Round2-var31
UGTSL2_S255C_V270I_I333L 81% 2.87 Round2-var32
UGTSL2_R6H_H247P_A341V 119% 2.05 Round2-var33
UGTSL2_H247P_R312L_G387E 67% Nd Round2-var34 UGTSL2_R6H_I240L_T329V
0% Nd Round2-var35 UGTSL2_S258T_V270I_T392A 146% 1.71 Round2-var36
UGTSL2_F253Y_T329I_I333L 76% 1.69 Round2-var37
UGTSL2_S258T_A285V_A341V 187% 2.32 Round2-var38
UGTSL2_L276A_A285L_M354L 12% Nd Round2-var39
UGTSL2_Q27R_F253Y_T329V 35% Nd Round2-var40 UGTSL2_R6H_S258T_L276A
36% Nd Round2-var41 UGTSL2_S258T_N278G_M354L 88% 0.96 Round2-var42
UGTSL2_R6H_F253Y_A285L 72% 1.40 Round2-var43
UGTSL2_F253Y_R312L_I352V 8% Nd Round2-var44
UGTSL2_I240L_A285V_I333L 43% 1.06 Round2-var45
UGTSL2_S255C_N278G_T329V 45% 2.51 *Mutations are noted as follows:
reference gene-original amino acid-position-new amino acid: For
example the mutation of an isoleucine at position 240 to a Leucine
for UGTSL2 is noted as UGTSL2 (I240L). Nd means Not determined.
[0696] Modeling of these results allowed to obtain a ranking of the
effect of each mutation. The following mutations were determined as
being beneficial for activity:
[0697] N325S, G387E, A285V, I333L, V270I, Q27R, N278G, L393V,
S258T, A341V, H247P and T392A.
[0698] The following mutations were determined as being beneficial
for an improved ratio between initial rate for the conversion of
Rebaudioside A and Rebaudioside D:
[0699] V270I, T392A, T329V, L276A, L393V, A341V and S255C.
Example 49
[0700] Use of .beta.-glucosidases for the conversion of
Rebaudioside M2 to Rebaudioside D Different .beta.-glucosidases
were tested for the hydrolysis of Rebaudioside M2. The goal was to
selectively hydrolyze the (1.fwdarw.6) glucoside bond in order to
obtain Rebaudioside D. The desired general reaction scheme is as
follows:
##STR00019##
[0701] First the selected .beta.-glucosidases were tested on
reference substrate 4-nitrophenyl-.beta.-D-glucopyranoside to
determine the activity. Based on the determined activities, the
used quantities of enzyme were calculated as units for use in the
hydrolysis of Rebaudioside M2.
[0702] The tested .beta.-glucosidases are depicted in the following
table:
TABLE-US-00076 Activity enzyme using 4- Quantity of enzyme used
nitrophenyl-.beta.-D-glucopyranoside (mg/mL of reaction
.beta.-glucosidase Manufacturer* (mmol min.sup.-1 mg.sup.-1)
mixture)/(U/mL) Isolase NEC 0.29 1.50/0.44 Aromase Amano 0.030
11.3/0.35 Naringinase Amano 0.017 13.5/0.23 Cellulase Tr
(Celluclast .RTM.1.5L) Novozyme 0.026 20.4/0.53 Cellobiase As
(Novozyme 188) Novozyme 0.26 1.50/0.39 CWD (Viscozyme .RTM.L)
Novozyme 0.0062 132/0.82 *Isolase (011410; National Enzyme Company,
USA); Aromase (GLY0151441; Amano Enzyme, Japan); Naringinase
(NAH0550102; Amano Enzyme, Japan), Cellulase from Trichoderma
reesei ATCC 26921 (Sigma C2730); Cellobiase from Aspergillus niger
(Sigma C6105); Viscozyme L (Sigma V2010)
[0703] The assay conditions were as follows:
[0704] Reactions were performed at 30.degree. C. at a total volume
of 10 mL containing 15 mM of sodium acetate buffer (pH 4.5) and 1
mM Rebaudioside M2. The reaction was started by the addition of
enzyme.
[0705] 625 .mu.L of reaction mixture was sampled after 0, 0.5, 1,
1.5, 2, 2.5, 3 and 3.3 hrs and quenched with a mixture of 575 .mu.L
of 80% methanol and 50 .mu.L of 2N H.sub.2SO.sub.4. The samples
were analyzed by HPLC analysis (CAD detection) using the analytical
method that was described above.
[0706] The reaction profiles of these reactions with different
.beta.-glucosidases are shown in FIGS. 68a-f.
[0707] It can be concluded that Naringinase and CWD catalyzed the
formation of Rebaudioside D2 and Rebaudioside A which indicates a
(1.fwdarw.2) bond glucolysis and (1.fwdarw.6) bond glucolysis
respectively. These enzymes can be considered as non-selective for
the conversion of Rebaudioside M2.
[0708] Isolase, Cellulase Tr and Cellobiase As possess a clear-cut
selectivity for the conversion of Rebaudioside M2 to Rebaudioside D
(hydrolysis of (1.fwdarw.6) glucoside bond), whereas Aromase
possessed low overall activity for the conversion of Rebaudioside
M2.
Example 50
[0709] Stability of Rebaudiosides in the presence of Isolase,
Cellulase Tr and Cellobiase As
[0710] In order to assess the selectivity of Isolase, Cellulase Tr
and Cellobiase As for Rebaudioside M2, Rebaudioside A, Rebaudioside
D and Rebaudioside M were tested as substrates under the following
conditions:
[0711] Reactions were performed over 24 h. at 30.degree. C. at a
total volume of 10 mL containing 15 mM of sodium acetate buffer (pH
4.5) and 1 mM of Rebaudioside A, Rebaudioside D or Rebaudioside M.
The reaction was started by the addition of enzyme.
[0712] 625 .mu.L of reaction mixture was sampled after 0, 0.5, 1,
1.5, 2, 2.5, 3 and 3.3 hrs and quenched with a mixture of 575 .mu.L
of 80% methanol and 50 .mu.L of 2N H.sub.2SO.sub.4. The samples
were analyzed by HPLC.
[0713] The results shown in FIGS.> 69a-c were obtained. It can
be observed that no significant conversion of Rebaudioside A,
Rebaudioside D and Rebaudioside M can be observed in the presence
of Isolase, Cellulase Tr and Cellobiase As.
Example 51
[0714] Four-Enzyme Reaction for the Conversion of Rebaudioside A to
Rebaudioside M
[0715] The influence of adding Isolase, Cellulase Tr or Cellobiase
As to the one-pot reaction of Rebaudioside A to Rebaudioside M with
UGTSL2, UGT76G1-1R-F12 and AtSUS was studied. The following
reaction conditions were used:
TABLE-US-00077 Compound Assay conc (mM) Rebaudioside A 2 UDP 0.25
MgCl.sub.2 3 Phosphate buffer pH 7.0 50 Sucrose 100 UGTSL2 lysate
(2.1 U/mL) 25 .mu.L/mL (0.053 U/mL) UGT76G1-R1-F12 lysate (2.5
U/mL) 170 .mu.L/mL (0.425 U/mL) AtSUS (130 U/mL) 25 .mu.L/mL (3.25
U/mL) Isolase 0.3 mg/mL Cellulase Tr 0.3 mg/mL Cellobiase As 4.0
mg/mL
[0716] The results for the experiments without and with added
.beta.-glucosidase are shown in FIGS. 70a-d. It can be seen that
addition of Cellobiase As is blocking the reaction and that
addition of Cellulase Tr does not have an influence on the reaction
profile. However, addition of Isolase to the reaction mixture has a
positive effect on the quantity of Rebaudioside M that is formed in
the reaction. An increase of almost 20% is observed when Isolase is
added. The Rebaudioside M2 content is approximately 10% lower and
Rebaudioside I content is approximately 15% lower when Isolase is
added to the reaction mixture compared to the reaction without
added .beta.-glucosidase.
[0717] Further improvement Reb M yield and reduction of Reb M2 and
Reb I content can be achieved through optimization of the reaction
parameters and amount of Isolase.
Example 52
[0718] Use of .beta.-glucosidases for the conversion of
Rebaudioside I to Rebaudioside A
[0719] Three .beta.-glucosidases were tested for the hydrolysis of
Rebaudioside I to Rebaudioside A. The goal was to selectively
hydrolyze the (1.fwdarw.6) glucoside bond in order to obtain
Rebaudioside D. The desired general reaction scheme is as
follows:
##STR00020##
[0720] The selected .beta.-glucosidases were tested on reference
substrate 4-nitrophenyl-.beta.-D-glucopyranoside to determine the
activity. Based on the determined activities, the used quantities
of enzyme were calculated as units for use in the hydrolysis of
Rebaudioside I. The tested .beta.-glucosidases are depicted in the
following table:
TABLE-US-00078 Activity enzyme using 4- nitrophenyl-.beta.-D-
Quantity of enzyme used glucopyranoside (mg/mL of reaction
.beta.-glucosidase Manufacturer* (mmol min.sup.-1 mg.sup.-1)
mixture)/(U/mL) Isolase NEC (011410) 0.29 1.50/0.44 Cellulase Tr
(Celluclast .RTM.1.5L) Novozymes* 0.026 22.3/0.58 Cellobiase As
(Novozyme 188) Novozymes* 0.26 1.60/0.42 *Isolase (011410; National
Enzyme Company, USA); Cellulase from Trichoderma reesei ATCC 26921
(Sigma C2730); Cellobiase from Aspergillus niger (Sigma C6105)
[0721] The assay conditions were as follows. Reactions were
performed at 30.degree. C. at a total volume of 2 mL containing 15
mM of sodium acetate buffer (pH 4.5) and 1 mM Rebaudioside I. The
reaction was started by the addition of enzyme. 125 .mu.L of
reaction mixture was sampled after 0, 1.5, 2.5 and 18 h. and
quenched with a mixture of 115 .mu.L of 80% methanol and 10 .mu.L
of 2N H.sub.2SO.sub.4. The samples were analyzed by HPLC analysis
(CAD detection) using the analytical method that was described
above. The reaction profiles of the different .beta.-glucosidases
with Rebaudioside I are depicted in the graph shown in FIG. 71.
[0722] It can be observed that all three tested .beta.-glucosidases
converted Rebaudioside I. The sole product was Rebaudioside A.
Example 53
[0723] Directed evolution of UGTSL2 for the conversion of
Rebaudioside A to Rebaudioside D (Round 3)
[0724] Taking the native enzyme UGTSL2 (GI_460410132) as baseline,
a list of 13 mutations that were identified during round 2 (EXAMPLE
48) and another list of 12 new mutations that were obtained by
DNA2.0 ProteinGPS.TM. strategy were prepared. This list of
mutations was subsequently used to design 46 variant genes that
contained each 1 to 8 different mutations. After codon-optimized
for expression in E. coli the genes were synthesized, subcloned in
the pET30a+ plasmid and used for transformation of E. coli BL21
(DE3) chemically competent cells. The obtained cells were grown in
Petri-dishes on solid LB medium in the presence of Kanamycin.
Suitable colonies were selected and allowed to grow in liquid LB
medium in tubes. Glycerol was added to the suspension as
cryoprotectant and 400 .mu.L aliquots were stored at -20.degree. C.
and at -80.degree. C.
[0725] These storage aliquots of E. coli BL21(DE3) containing the
pET30a+_UGTSL2var plasmids were thawed and added to LBGKP medium
(20 g/L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM
Phosphate buffer pH 7.00; 2.5 g/L glucose and 50 mg/L of
Kanamycin). This culture was allowed to shake in a 96 microtiter
plate at 30.degree. C. for 8 h.
[0726] 3.95 mL of production medium containing 60 g/L of Overnight
Express.TM. Instant TB medium (Novagen.RTM.), 10 g/L of glycerol
and 50 mg/L of Kanamycin was inoculated with 50 .mu.L of above
described culture. In a 48 deepwell plate the resulting culture was
allowed to stir at 20.degree. C. The cultures gave significant
growth and a good OD (600 nm) was obtained. After 44 h, the cells
were harvested by centrifugation and frozen.
[0727] Lysis was performed by addition of Bugbuster.RTM. Master mix
(Novagen.RTM.) to the thawed cells and the lysates were recovered
by centrifugation.
[0728] In order to measure the activity of the variants for the
transformation of Rebaudioside A to Rebaudioside D, 100 .mu.L of
fresh lysate was added to a solution of Rebaudioside A (final
concentration 0.5 mM), MgCl.sub.2 (final concentration 3 mM) and
UDP-Glucose (final concentration 2.5 mM) in 50 mM phosphate buffer
pH 7.2. The reaction was allowed to run at 30.degree. C. and
samples were taken after 2, 4, 6 and 22 h. to determine the initial
rates after HPLC analysis (CAD detection) using the analytical
method that was described above for the transformation of
Rebaudioside A to Rebaudioside D.
[0729] In parallel 100 .mu.L of fresh lysate was added to a
solution of Rebaudioside D (final concentration 0.5 mM), MgCl.sub.2
(final concentration 3 mM) and UDP-Glucose (final concentration 2.5
mM) in 50 mM phosphate buffer pH 7.2. The reaction was allowed to
run at 30.degree. C. and samples were taken after 2, 4, 6 and 22 h.
to determine the initial rates for Rebaudioside D conversion after
HPLC analysis (CAD detection).
[0730] Apart from the new variants for this round, both experiments
were also performed with baseline clone, UGTSL2. The initial rates
for the conversion of Rebaudioside A or Rebaudioside D for this
baseline clone were defined as 100%.
[0731] Activity of each clone was defined as normalized activity
compared to baseline clone UGTSL2 whereas specificity of each clone
was expressed as the ratio between the initial rates for the
conversion of Rebaudioside A and Rebaudioside D.
[0732] The normalized initial rate for the conversion of
Rebaudioside A and the ratio between the initial rates for the
conversion of Rebaudioside A and Rebaudioside D are depicted in the
following table.
TABLE-US-00079 Ratio between initial Normalized initial rates for
the rate for conversion of conversion of Rebaudioside A and Clone
Mutations* Rebaudioside A Rebaudioside D UGTSL2 Baseline clone 100%
1.67 Round3-var1 UGTSL2_S255C_A285V_V349L_L393V 13% 1.86
Round3-var2 UGTSL2_N130G_S255C_N339G_T392A 264% 3.09 Round3-var3
UGTSL2_S255C_V270I_L276A_A285V 10% 4.50 Round3-var4
UGTSL2_S255C_A285I_T329V_H357Y_T392A 70% 4.87 Round3-var5
UGTSL2_S255C_A341V_T392A_I412M 359% 4.34 Round3-var6
UGTSL2_S255C_A285V_K301E_A341V_T392A_L393V 104% 4.34 Round3-var7
UGTSL2_S255C_L276A_K301E_T392A 79% 4.51 Round3-var8
UGTSL2_S255C_T392A_L393V_I412L 46% 2.12 Round3-var9
UGTSL2_F226V_S255C_V270I_T392A 226% 2.67 Round3-var10
UGTSL2_S255C_L276A_A285V_T329V_T392A_I412L 5% 8.57 Round3-var11
UGTSL2_S255C_H357Y_T392A_K408R 0% Nd Round3-var12
UGTSL2_S255C_V270I_A285V_A341V_T392A_I412L 403% 7.83 Round3-var13
UGTSL2_S255C_A285V_T329V_N339G_A341V_V349L_T392A 0% Nd Round3-var14
UGTSL2_N130G_A285V_A341V_T392A_K408R 475% 2.69 Round3-var15
UGTSL2_T329V 122% 2.62 Round3-var16
UGTSL2_P225L_F226V_S255C_A285V_T329V_T392A_L393V 14% 3.03
Round3-var17 UGTSL2_I203L_P225L_S255C_V349L_T392A 0% Nd
Round3-var18 UGTSL2_V270I_A285I_K301E_T392A 390% 1.40 Round3-var19
UGTSL2_I203L_S255C_V270I_A285V_N339G_T392A_L393V 12% 1.81
Round3-var20 UGTSL2_N130G_S255C_L276A_A285I_T392A_L393V 262% 3.35
Round3-var21 UGTSL2_S255C_V270I_A285V_T329V_T392A_K408R_I412M 67%
3.33 Round3-var22 UGTSL2_I203L_F226V_S255C_L276A_A285V_T392A_I412M
0% Nd Round3-var23 UGTSL2_P225L_S255C_L276A_A285V_A341V_H357Y_T392A
1% Nd Round3-var24 UGTSL2_S258T_K408R 58% 3.12 Round3-var25
UGTSL2_F226V_H247P_S258T_A341V 85% 2.47 Round3-var26
UGTSL2_S258T_V270I_A341V_V349L 5% 1.74 Round3-var27
UGTSL2_S258T_L276A_A285V_K301E_A341V_L393V 297% 2.26 Round3-var28
UGTSL2_P225L_S258T_L276A_A341V 22% 1.08 Round3-var29
UGTSL2_S258T_L276A_N339G_A341V 18% 1.08 Round3-var30
UGTSL2_S258T_V270I_N278G_A285V_A341V_T392A 313% 2.29 Round3-var31
UGTSL2_F253Y_A341V_L393V 105% 3.88 Round3-var32
UGTSL2_N130G_S258T_N278G_A341V_H357Y 13% 1.66 Round3-var33
UGTSL2_H247P_S258T_N278G_A285V_A341V_L393V_K408R 286% 3.29
Round3-var34 UGTSL2_F253Y_S258T_V270I_L276A_A285I_A341V 362% 1.90
Round3-var35 UGTSL2_F253Y_S255C_S258T_A341V_T392A 24% 3.28
Round3-var36 UGTSL2_S255C_S258T_L276A_N278G_A285V_I333L_A341V 121%
3.36 Round3-var37 UGTSL2_F226V_S258T_I333L 5% 1.20 Round3-var38
UGTSL2_S255C_S258T_V270I_A285V_T329V 139% 2.59 Round3-var39
UGTSL2_S258T_L276A_A285V_H357Y_T392A 94% 1.98 Round3-var40
UGTSL2_S258T_N278G_K301E_T329V_A341V_I412L 179% 2.82 Round3-var41
UGTSL2_P225L_S258T_A285I_L393V_I412L 1% 0.59 Round3-var42
UGTSL2_I203L_N278G_A285V_I412M 3% 2.68 Round3-var43
UGTSL2_I203L_S258T_V270I_I333L_A341V_L393V 44% 6.27 Round3-var44
UGTSL2_S258T_A285V_T329V_N339G_A341V_V349L_T392A_L393V 0% Nd
Round3-var45 UGTSL2_N130G_H247P_V270I_A285V_A341V_T392A 869% 2.69
Round3-var46 UGTSL2_S258T_A341V_T392A_I412M 132% 3.27 *Mutations
are noted as follows: reference gene-original amino
acid-position-new amino acid: For example the mutation of an
isoleucine at position 240 to a Leucine for UGTSL2 is noted as
UGTSL2 (I240L). Nd means Not determined.
[0733] Modeling of these results allowed to obtain a ranking of the
effect of each mutation. The following mutations were determined as
being beneficial for activity:
[0734] N130G, H247P, F253Y, V270I, L276A, A285I, A285V, K301E,
A341V, T392A, K408R, I412L.
[0735] The following mutations were determined as being beneficial
for an improved ratio between initial rate for the conversion of
Rebaudioside A and Rebaudioside D:
[0736] I203L, S255C, I333L, A341V, H357Y, L393V, K408R, I412L.
Example 54
[0737] One-pot, four-enzyme conversion of Rebaudioside A to
Rebaudioside M 10 mL of a reaction mixture containing 5.0 mM of
Rebaudioside A, 0.25 mM of UDP, 2 mM of MgCl.sub.2, 100 mM of
sucrose, 50 mM of potassium phosphate buffer pH 7.5, 2.5 U of
UGTSL2-R3-D2 (UGTSL2-Round3-var12, see EXAMPLE 53), 25 U of
UGT76G1-R3-G3 (UGT76G1-Round3-var21, see EXAMPLE 44), 25 U of AtSUS
and 5 U of Isolase.RTM. was filtered through a 0.2 .mu.m filter in
a sterile flask. The resulting reaction mixture was gently shaken
at 30.degree. C. for 65 h.
[0738] Samples were taken under sterile conditions at regular
intervals by taking 125 .mu.l, of reaction mixture and quenching it
with 10 .mu.L of 2 N H.sub.2SO.sub.4 and 765 .mu.l, of 50%
methanol. After centrifugation, 200 .mu.L of the supernatant was
analyzed by HPLC.
[0739] The reaction profile shown in FIG. 72a was obtained. The
HPLC analysis after 48 h of reaction is shown in FIG. 72b.
Example 55
[0740] One-pot, four-enzyme conversion of Rebaudioside A to
Rebaudioside M
[0741] 10 mL of a reaction mixture containing 10.0 mM of
Rebaudioside A, 0.50 mM of UDP, 3 mM of MgCl.sub.2, 100 mM of
sucrose, 50 mM of potassium phosphate buffer pH 7.5, 5.0 U of
UGTSL2-R3-D2 (UGTSL2-Round3-var12, see EXAMPLE 53), 50 U of
UGT76G1-R3-G3 (UGT76G1-Round3-var21, see EXAMPLE 44), 50 U of AtSUS
and 10 U of Isolase.RTM. was filtered through a 0.2 .mu.m filter in
a sterile flask. The resulting reaction mixture was gently shaken
at 30.degree. C. for 66 h.
[0742] Samples were taken under sterile conditions at regular
intervals by taking 125 .mu.L of reaction mixture and quenching it
with 10 .mu.L of 2N H.sub.2SO.sub.4 and 765 .mu.L of 50% methanol.
After centrifugation, 200 .mu.L of the supernatant was analyzed by
HPLC.
[0743] The reaction profile shown in FIG. 73a was obtained. The
HPLC analysis after 48 h of reaction is shown in FIG. 73b.
Example 56
[0744] One-Pot, Four-Enzyme Conversion of Rebaudioside a to
Rebaudioside M
[0745] 50 mL of a reaction mixture containing 10.0 mM of
Rebaudioside A, 0.5 mM of UDP, 4 mM of MgCl.sub.2, 100 mM of
sucrose, 50 mM of potassium phosphate buffer pH 7.5, 25 U of
UGTSL2-R3-D2 (UGTSL2-Round3-var12, see EXAMPLE 53), 250 U of
UGT76G1-R3-G3 (UGT76G1-Round3-var21, see EXAMPLE 44), 250 U of
AtSUS and 50 U of Isolase.RTM. was filtered through a 0.2 .mu.m
filter in a sterile flask. The resulting reaction mixture was
gently shaken at 35.degree. C. for 95 hrs.
[0746] Samples were taken under sterile conditions at regular
intervals by taking 125 .mu.L of reaction mixture and quenching it
with 10 .mu.L of 2 N.sub.2SO.sub.4 and 765 .mu.L of 50% methanol.
After centrifugation, 200 .mu.L of the supernatant was analyzed by
HPLC.
[0747] At the end of the reaction, the reaction mixture became a
fine suspension. Filtration of the suspension and HPLC analysis of
the residue and filtrate showed that the Reb M content in the
filtrate was 79% and that the Reb M content in the solid was
97%.
[0748] The reaction profile shown in FIG. 74a was obtained. The
HPLC of the reaction mixture after 95 hrs is shown in FIG. 74b.
Example 57
[0749] One-pot, four-enzyme conversion of Rebaudioside A to
Rebaudioside M (addition of UGT76G1 and Isolase after 6.5 h)
[0750] A reaction mixture containing Rebaudioside A, UDP,
MgCl.sub.2, sucrose, potassium phosphate buffer pH 7.5,
UGTSL2-R3-D2 (UGTSL2-Round3-var12, see EXAMPLE 53) and AtSUS was
filtered through a 0.2 .mu.m filter in a sterile flask. The
resulting reaction mixture was gently shaken at 35.degree. C. for
6.5 h. UGT76G1-R3-G3 (UGT76G1-Round3-var21, see EXAMPLE 44) and
Isolase.RTM. were added and the reaction mixture was filtered
through a 0.2 .mu.m filter in a sterile flask and gently shaken for
another 89 h at 35.degree. C. The final volume of the reaction
mixture was 50 mL and final concentrations of reagents and enzymes
were as follows: 10.0 mM of Rebaudioside A, 0.5 mM of UDP, 4 mM of
MgCl.sub.2, 100 mM of sucrose, 50 mM of potassium phosphate buffer
pH 7.5, 25 U of UGTSL2-R3-D2, 250 U of UGT76G1-R3-G3, 250 U of
AtSUS and 50 U of Isolase.RTM. Samples were taken under sterile
conditions at regular intervals by taking 125 .mu.L of reaction
mixture and quenching it with 10 .mu.L of 2 N H.sub.2SO.sub.4 and
765 .mu.L of 50% methanol. After centrifugation, 200 .mu.L of the
supernatant was analyzed by HPLC.
[0751] The reaction profile shown in FIG. 75a was obtained. The
HPLC of the reaction mixture after 95 h is shown in FIG. 75b.
Example 58
[0752] One-pot, four-enzyme conversion of Rebaudioside A to
Rebaudioside M (addition of UGT76G1 and Isolase after 6.5 h)
[0753] A reaction mixture containing Rebaudioside A, UDP,
MgCl.sub.2, sucrose, potassium phosphate buffer pH 7.5,
UGTSL2-R3-D2 (UGTSL2-Round3-var12, see EXAMPLE 53) and AtSUS was
filtered through a 0.2 .mu.m filter in a sterile flask. The
resulting reaction mixture was gently shaken at 35.degree. C. for
6.5 h. UGT76G1-R3-G3 (UGT76G1-Round3-var21, see EXAMPLE 44) and
Isolase.RTM. were added and the reaction mixture was filtered
through a 0.2 .mu.m filter in a sterile flask and gently shaken for
another 89 h at 35.degree. C. The final volume of the reaction
mixture was 50 mL and the final concentrations of reagents and
enzymes were as follows: 10.0 mM of Rebaudioside A, 0.5 mM of UDP,
4 mM of MgCl.sub.2, 100 mM of sucrose, 50 mM of potassium phosphate
buffer pH 7.5, 25 U of UGTSL2-R3-D2, 250 U of UGT76G1-R3-G3, 250 U
of AtSUS and 25 U of Isolase.RTM..
[0754] Samples were taken under sterile conditions at regular
intervals by taking 125 .mu.L of reaction mixture and quenching it
with 10 .mu.L of 2 N H.sub.2SO.sub.4 and 765 .mu.L of 50% methanol.
After centrifugation, 200 .mu.L of the supernatant was analyzed by
HPLC.
[0755] At the end of the reaction, the reaction mixture became a
fine suspension. Filtration of the suspension and HPLC analysis of
the residue and filtrate showed that the Reb M content in the
filtrate was 81% and that the Reb M content in the solid was
98%.
[0756] The reaction profile shown in FIG. 76a was obtained. The
HPLC of the reaction mixture after 95 h is shown in FIG. 76b.
Example 59
[0757] Directed evolution of UGTSL2 for the conversion of
Rebaudioside A to Rebaudioside D (Round 4)
[0758] The most active enzyme from the third round (see EXAMPLE 53)
UGTSL2_round3-var45 was taken as starting point. The five best
mutations for activity from round 3 were used to create a set of 10
variants containing each two of these mutations. After
codon-optimized for expression in E. coli the genes were
synthesized, subcloned in the pET30a+ plasmid and used for
transformation of E. coli BL21 (DE3) chemically competent cells.
The obtained cells were grown in Petri-dishes on solid LB medium in
the presence of Kanamycin. Suitable colonies were selected and
allowed to grow in liquid LB medium in tubes. Glycerol was added to
the suspension as cryoprotectant and 400 .mu.L aliquots were stored
at -20.degree. C. and at -80.degree. C.
[0759] These storage aliquots of E. coli BL21(DE3) containing the
pET30a+_UGTSL2var plasmids were thawed and added to LBGKP medium
(20 g/L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM
Phosphate buffer pH 7.00; 2.5 g/L glucose and 50 mg/L of
Kanamycine). This culture was allowed to shake in a 96 microtiter
plate at 30.degree. C. for 8 h. 3.95 mL of production medium
containing 60 g/L of Overnight Express.TM. Instant TB medium
(Novagen.RTM.), 10 g/L of glycerol and 50 mg/L of Kanamycin was
inoculated with 50 .mu.L of above described culture. In a 48
deepwell plate the resulting culture was allowed to stir at
20.degree. C. The cultures gave significant growth and a good OD
(600 nm) was obtained. After 44 h, the cells were harvested by
centrifugation and frozen. Lysis was performed by addition of
Bugbuster.RTM. Master mix (Novagen.RTM.) to the thawed cells and
the lysates were recovered by centrifugation. Lysates were diluted
five-fold with water before activity testing.
[0760] In order to measure the activity of the variants for the
transformation of Rebaudioside A to Rebaudioside D, 100 .mu.L of
fresh lysate was added to a solution of Rebaudioside A (final
concentration 0.5 mM), MgCl.sub.2 (final concentration 3 mM) and
UDP-Glucose (final concentration 2.5 mM) in 50 mM phosphate buffer
pH 7.2. The reaction was allowed to run at 30.degree. C. and
samples were taken after 2, 4, 6 and 22 h. to determine the
activities after HPLC analysis (CAD detection) using the analytical
method that was described above for the transformation of
Rebaudioside A to Rebaudioside D.
[0761] Selectivity of each clone was determined by measuring the
amount of Rebaudioside M2 that was formed at 100% UDP-Glc
conversion (defined as (2*[Reb M2]+[Reb D])/([Reb A]+[Reb D]+[Reb
M2]).
[0762] In parallel the experiments were performed with baseline
clone, UGTSL2-Round3-Var45. The initial rate for this baseline
clone was defined as 100%. The relative initial rates and the
amounts of Rebaudioside M2 that are formed at 100% UDP-Glc
conversion for the round 4 clones are depicted in the following
table:
TABLE-US-00080 Normalized initial rate for Rebaudioside M2 content
at Clone Mutations* conversion of Rebaudioside A 100% UDP-Glc
conversion Round3-var45 UGTSL2 100% 15.80%
(N130G_H247P_V270I_A285V_A341V_T392A) Round4-var1
UGTSL2-Round3-var45 (K301E_V285I) 96% 15.90% Round4-var2
UGTSL2-Round3-var45 (K301E_I412L) 90% 15.30% Round4-var3
UGTSL2-Round3-var45 (K301E_L276A) 135% 16.80% Round4-var4
UGTSL2-Round3-var45 (K301E_K408R) 90% 14.90% Round4-var5
UGTSL2-Round3-var45 (V285I_I412L) 77% 15.60% Round4-var6
UGTSL2-Round3-var45 (V285I_L276A) 124% 16.60% Round4-var7
UGTSL2-Round3-var45 (V285I_K408R) 98% 16.50% Round4-var8
UGTSL2-Round3-var45 (I412L_L276A) 88% 15.10% Round4-var9
UGTSL2-Round3-var45 (I412L_K408R) 82% 15.00% Round4-var10
UGTSL2-Round3-var45 (L276A_K408R) 93% 15.40% *Mutations are noted
as follows: reference gene-original amino acid-position-new amino
acid: For example the mutation of an isoleucine at position 240 to
a Leucine for UGTSL2 is noted as UGTSL2 (I240L).
Example 60
[0763] Directed evolution of UGT76G1 for the conversion of
Rebaudioside D to Rebaudioside X (Round 4)
[0764] The most active clone from the third round of directed
evolution of UGT76G1 (see EXAMPLE 44 round3_UGT76G1var21 containing
mutations: I46L_K303G_K393R) was chosen as baseline clone for round
4. The best identified mutations from round 3 (S119A, 274G, 1295M,
F314S and K334R) were used to create a set of 10 variants that
contained each 2 of these mutations. After codon-optimized for
expression in E. coli the genes were synthesized, subcloned in the
pET30a+ plasmid and used for transformation of E. coli BL21 (DE3)
chemically competent cells. The obtained cells were grown in
Petri-dishes on solid LB medium in the presence of Kanamycin.
Suitable colonies were selected and allowed to grow in liquid LB
medium in tubes. Glycerol was added to the suspension as
cryoprotectant and 400 .mu.L aliquots were stored at -20.degree. C.
and at -80.degree. C. These storage aliquots of E. coli BL21(DE3)
containing the pET30a+_UGT76G1var plasmids were thawed and added to
LBGKP medium (20 g/L Luria Broth Lennox; 50 mM PIPES buffer pH
7.00; 50 mM Phosphate buffer pH 7.00; 2.5 g/L glucose and 50 mg/L
of Kanamycine). This culture was allowed to shake in a 96
microtiter plate at 30.degree. C. for 8 h. 3.95 mL of production
medium containing 60 g/L of Overnight Express.TM. Instant TB medium
(Novagen.RTM.), 10 g/L of glycerol and 50 mg/L of Kanamycin was
inoculated with 50 .mu.L of above described culture. In a 48
deepwell plate the resulting culture was allowed to stir at
20.degree. C. The cultures gave significant growth and a good OD
(600 nm) was obtained. After 44 h, the cells were harvested by
centrifugation and frozen.
[0765] Lysis was performed by addition of Bugbuster.RTM. Master mix
(Novagen.RTM.) to the thawed cells and the lysate was recovered by
centrifugation. Activity tests were performed with 100 .mu.L of
fresh lysate that was added to a solution of Rebaudioside D (final
concentration 0.5 mM), MgCl2 (final concentration 3 mM) and
UDP-Glucose (final concentration 2.5 mM) in 50 mM phosphate buffer
pH 7.2.
[0766] The reaction was allowed to run at 30.degree. C. and samples
were taken after 1, 2, 4, 6 and 22 h. to determine conversion and
initial rate by HPLC (CAD detection) using the analytical method
that was described above for the transformation of Rebaudioside D
to Rebaudioside X. In parallel the experiments were performed with
baseline clone, Round3-Var21. The conversion after 22 h. and
initial rate for this baseline clone was defined as 100% and the
normalized conversions and initial rates for the round 4 clones are
depicted in the following table:
TABLE-US-00081 Normalized Normalized conversion Reb D initial Clone
Mutations* to Reb X after 22 h. rate (0-4 h) Round3-Var21 UGT76G1
100% 100%
(S42A_F46L_Q266E_P272A_K303G_R334K_G348P_L379G_K393R_I407V)
Round4-Var1 Round3-Var21 (S119A_S274G) 99.5% 100% Round4-Var2
Round3-Var21 (S119A_I295M) 95.4% 93% Round4-Var3 Round3-Var21
(S119A_F314S) 87.5% 77% Round4-Var4 Round3-Var21 (S119A_K334R)
94.0% 81% Round4-Var5 Round3-Var21 (S274G_I295M) 88.8% 77%
Round4-Var6 Round3-Var21 (S274G_F314S) 86.7% 75% Round4-Var7
Round3-Var21 (S274G_K334R) 89.8% 74% Round4-Var8 Round3-Var21
(I295M_F314S) 84.3% 72% Round4-Var9 Round3-Var21 (I295M_K334R)
81.2% 60% Round4-Var10 Round3-Var21 (F314S_K334R) 85.6% 74%
*Mutations are noted as follows: reference gene-original amino
acid-position-new amino acid: For example the mutation of Serine at
position 119 to Alanine for variant 1 from the fourth round of
directed evolution of UGT76G1 is noted as Round3-Var21 (S119A)
[0767] It is to be understood that the foregoing descriptions and
specific embodiments have fully disclosed, illustrated and enabled
the best mode of the invention and the principles thereof, and that
modifications and additions may be made by those skilled in the art
without departing from the spirit and scope of the invention, which
is limited only by the scope of the appended claims.
Sequence CWU 1
1
1311397DNAStevia rebaudiana 1ccatggccca tatggaaaac aaaaccgaaa
ccaccgttcg tcgtcgtcgc cgtattattc 60tgtttccggt tccgtttcag ggtcatatta
atccgattct gcagctggca aatgtgctgt 120atagcaaagg ttttagcatt
accatttttc ataccaattt taacaaaccg aaaaccagca 180attatccgca
ttttaccttt cgctttattc tggataatga tccgcaggat gaacgcatta
240gcaatctgcc gacacatggt ccgctggcag gtatgcgtat tccgattatt
aacgaacatg 300gtgcagatga actgcgtcgt gaactggaac tgctgatgct
ggcaagcgaa gaagatgaag 360aagttagctg tctgattacc gatgcactgt
ggtattttgc acagagcgtt gcagatagcc 420tgaatctgcg tcgtctggtt
ctgatgacca gcagcctgtt taactttcat gcacatgtta 480gcctgccgca
gtttgatgaa ctgggttatc tggatccgga tgataaaacc cgtctggaag
540aacaggcaag cggttttccg atgctgaaag tgaaagatat caaaagcgcc
tatagcaatt 600ggcagattct gaaagaaatt ctgggcaaaa tgattaaaca
gaccaaagca agcagcggtg 660ttatttggaa tagctttaaa gaactggaag
aaagcgaact ggaaaccgtg attcgtgaaa 720ttccggcacc gagctttctg
attccgctgc cgaaacatct gaccgcaagc agcagcagcc 780tgctggatca
tgatcgtacc gtttttcagt ggctggatca gcagcctccg agcagcgttc
840tgtatgttag ctttggtagc accagcgaag ttgatgaaaa agattttctg
gaaattgccc 900gtggtctggt tgatagcaaa cagagctttc tgtgggttgt
tcgtccgggt tttgttaaag 960gtagcacctg ggttgaaccg ctgccggatg
gttttctggg tgaacgtggt cgtattgtta 1020aatgggttcc gcagcaagaa
gttctggcac acggcgcaat tggtgcattt tggacccata 1080gcggttggaa
tagcaccctg gaaagcgttt gtgaaggtgt tccgatgatt tttagcgatt
1140ttggtctgga tcagccgctg aatgcacgtt atatgagtga tgttctgaaa
gtgggtgtgt 1200atctggaaaa tggttgggaa cgtggtgaaa ttgcaaatgc
aattcgtcgt gttatggtgg 1260atgaagaagg tgaatatatt cgtcagaatg
cccgtgttct gaaacagaaa gcagatgtta 1320gcctgatgaa aggtggtagc
agctatgaaa gcctggaaag tctggttagc tatattagca 1380gcctgtaata actcgag
139721442DNAStevia rebaudiana 2ccatggcaca tatggcaacc agcgatagca
ttgttgatga tcgtaaacag ctgcatgttg 60caacctttcc gtggctggca tttggtcata
ttctgccgta tctgcagctg agcaaactga 120ttgcagaaaa aggtcataaa
gtgagctttc tgagcaccac ccgtaatatt cagcgtctga 180gcagccatat
tagtccgctg attaatgttg ttcagctgac cctgcctcgt gttcaagaac
240tgccggaaga tgccgaagca accaccgatg ttcatccgga agatattccg
tatctgaaaa 300aagcaagtga tggtctgcag ccggaagtta cccgttttct
ggaacagcat agtccggatt 360ggatcatcta tgattatacc cattattggc
tgccgagcat tgcagcaagc ctgggtatta 420gccgtgcaca ttttagcgtt
accaccccgt gggcaattgc atatatgggt ccgagcgcag 480atgcaatgat
taatggtagt gatggtcgta ccaccgttga agatctgacc acccctccga
540aatggtttcc gtttccgacc aaagtttgtt ggcgtaaaca tgatctggca
cgtctggttc 600cgtataaagc accgggtatt agtgatggtt atcgtatggg
tctggttctg aaaggtagcg 660attgtctgct gagcaaatgc tatcatgaat
ttggcaccca gtggctgccg ctgctggaaa 720ccctgcatca ggttccggtt
gttccggtgg gtctgctgcc tccggaagtt ccgggtgatg 780aaaaagatga
aacctgggtt agcatcaaaa aatggctgga tggtaaacag aaaggtagcg
840tggtttatgt tgcactgggt agcgaagttc tggttagcca gaccgaagtt
gttgaactgg 900cactgggtct ggaactgagc ggtctgccgt ttgtttgggc
atatcgtaaa ccgaaaggtc 960cggcaaaaag cgatagcgtt gaactgccgg
atggttttgt tgaacgtacc cgtgatcgtg 1020gtctggtttg gaccagctgg
gcacctcagc tgcgtattct gagccatgaa agcgtttgtg 1080gttttctgac
ccattgtggt agcggtagca ttgtggaagg tctgatgttt ggtcatccgc
1140tgattatgct gccgattttt ggtgatcagc cgctgaatgc acgtctgctg
gaagataaac 1200aggttggtat tgaaattccg cgtaatgaag aagatggttg
cctgaccaaa gaaagcgttg 1260cacgtagcct gcgtagcgtt gttgttgaaa
aagaaggcga aatctataaa gccaatgcac 1320gtgaactgag caaaatctat
aatgatacca aagtggaaaa agaatatgtg agccagttcg 1380tggattatct
ggaaaaaaac acccgtgcag ttgccattga tcacgaaagc taatgactcg 1440ag
14423472PRTOryza sativa 3Met Asp Asp Ala His Ser Ser Gln Ser Pro
Leu His Val Val Ile Phe 1 5 10 15 Pro Trp Leu Ala Phe Gly His Leu
Leu Pro Cys Leu Asp Leu Ala Glu 20 25 30 Arg Leu Ala Ala Arg Gly
His Arg Val Ser Phe Val Ser Thr Pro Arg 35 40 45 Asn Leu Ala Arg
Leu Pro Pro Val Arg Pro Glu Leu Ala Glu Leu Val 50 55 60 Asp Leu
Val Ala Leu Pro Leu Pro Arg Val Asp Gly Leu Pro Asp Gly 65 70 75 80
Ala Glu Ala Thr Ser Asp Val Pro Phe Asp Lys Phe Glu Leu His Arg 85
90 95 Lys Ala Phe Asp Gly Leu Ala Ala Pro Phe Ser Ala Phe Leu Asp
Thr 100 105 110 Ala Cys Ala Gly Gly Lys Arg Pro Asp Trp Val Leu Ala
Asp Leu Met 115 120 125 His His Trp Val Ala Leu Ala Ser Gln Glu Arg
Gly Val Pro Cys Ala 130 135 140 Met Ile Leu Pro Cys Ser Ala Ala Val
Val Ala Ser Ser Ala Pro Pro 145 150 155 160 Thr Glu Ser Ser Ala Asp
Gln Arg Glu Ala Ile Val Arg Ser Met Gly 165 170 175 Thr Ala Ala Pro
Ser Phe Glu Ala Lys Arg Ala Thr Glu Glu Phe Ala 180 185 190 Thr Glu
Gly Ala Ser Gly Val Ser Ile Met Thr Arg Tyr Ser Leu Thr 195 200 205
Leu Gln Arg Ser Lys Leu Val Ala Met Arg Ser Cys Pro Glu Leu Glu 210
215 220 Pro Gly Ala Phe Thr Ile Leu Thr Arg Phe Tyr Gly Lys Pro Val
Val 225 230 235 240 Pro Phe Gly Leu Leu Pro Pro Arg Pro Asp Gly Ala
Arg Gly Val Ser 245 250 255 Lys Asn Gly Lys His Asp Ala Ile Met Gln
Trp Leu Asp Ala Gln Pro 260 265 270 Ala Lys Ser Val Val Tyr Val Ala
Leu Gly Ser Glu Ala Pro Met Ser 275 280 285 Ala Asp Leu Leu Arg Glu
Leu Ala His Gly Leu Asp Leu Ala Gly Thr 290 295 300 Arg Phe Leu Trp
Ala Met Arg Lys Pro Ala Gly Val Asp Ala Asp Ser 305 310 315 320 Val
Leu Pro Ala Gly Phe Leu Gly Arg Thr Gly Glu Arg Gly Leu Val 325 330
335 Thr Thr Arg Trp Ala Pro Gln Val Ser Ile Leu Ala His Ala Ala Val
340 345 350 Cys Ala Phe Leu Thr His Cys Gly Trp Gly Ser Val Val Glu
Gly Leu 355 360 365 Gln Phe Gly His Pro Leu Ile Met Leu Pro Ile Leu
Gly Asp Gln Gly 370 375 380 Pro Asn Ala Arg Ile Leu Glu Gly Arg Lys
Leu Gly Val Ala Val Pro 385 390 395 400 Arg Asn Asp Glu Asp Gly Ser
Phe Asp Arg Gly Gly Val Ala Gly Ala 405 410 415 Val Arg Ala Val Val
Val Glu Glu Glu Gly Lys Thr Phe Phe Ala Asn 420 425 430 Ala Arg Lys
Leu Gln Glu Ile Val Ala Asp Arg Glu Arg Glu Glu Arg 435 440 445 Cys
Ile Asp Glu Phe Val Gln His Leu Thr Ser Trp Asn Glu Leu Lys 450 455
460 Asn Asn Ser Asp Gly Gln Tyr Pro 465 470 4502PRTAvena strigosa
4Met Ala Val Lys Asp Glu Gln Gln Ser Pro Leu His Ile Leu Leu Phe 1
5 10 15 Pro Phe Leu Ala Pro Gly His Leu Ile Pro Ile Ala Asp Met Ala
Ala 20 25 30 Leu Phe Ala Ser Arg Gly Val Arg Cys Thr Ile Leu Thr
Thr Pro Val 35 40 45 Asn Ala Ala Ile Ile Arg Ser Ala Val Asp Arg
Ala Asn Asp Ala Phe 50 55 60 Arg Gly Ser Asp Cys Pro Ala Ile Asp
Ile Ser Val Val Pro Phe Pro 65 70 75 80 Asp Val Gly Leu Pro Pro Gly
Val Glu Asn Gly Asn Ala Leu Thr Ser 85 90 95 Pro Ala Asp Arg Leu
Lys Phe Phe Gln Ala Val Ala Glu Leu Arg Glu 100 105 110 Pro Phe Asp
Arg Phe Leu Ala Asp Asn His Pro Asp Ala Val Val Ser 115 120 125 Asp
Ser Phe Phe His Trp Ser Thr Asp Ala Ala Ala Glu His Gly Val 130 135
140 Pro Arg Leu Gly Phe Leu Gly Ser Ser Met Phe Ala Gly Ser Cys Asn
145 150 155 160 Glu Ser Thr Leu His Asn Asn Pro Leu Glu Thr Ala Ala
Asp Asp Pro 165 170 175 Asp Ala Leu Val Ser Leu Pro Gly Leu Pro His
Arg Val Glu Leu Arg 180 185 190 Arg Ser Gln Met Met Asp Pro Lys Lys
Arg Pro Asp His Trp Ala Leu 195 200 205 Leu Glu Ser Val Asn Ala Ala
Asp Gln Lys Ser Phe Gly Glu Val Phe 210 215 220 Asn Ser Phe His Glu
Leu Glu Pro Asp Tyr Val Glu His Tyr Gln Thr 225 230 235 240 Thr Leu
Gly Arg Arg Thr Trp Leu Val Gly Pro Val Ala Leu Ala Ser 245 250 255
Lys Asp Met Ala Gly Arg Gly Ser Thr Ser Ala Arg Ser Pro Asp Ala 260
265 270 Asp Ser Cys Leu Arg Trp Leu Asp Thr Lys Gln Pro Gly Ser Val
Val 275 280 285 Tyr Val Ser Phe Gly Thr Leu Ile Arg Phe Ser Pro Ala
Glu Leu His 290 295 300 Glu Leu Ala Arg Gly Leu Asp Leu Ser Gly Lys
Asn Phe Val Trp Val 305 310 315 320 Leu Gly Arg Ala Gly Pro Asp Ser
Ser Glu Trp Met Pro Gln Gly Phe 325 330 335 Ala Asp Leu Ile Thr Pro
Arg Gly Asp Arg Gly Phe Ile Ile Arg Gly 340 345 350 Trp Ala Pro Gln
Met Leu Ile Leu Asn His Arg Ala Leu Gly Gly Phe 355 360 365 Val Thr
His Cys Gly Trp Asn Ser Thr Leu Glu Ser Val Ser Ala Gly 370 375 380
Val Pro Met Val Thr Trp Pro Arg Phe Ala Asp Gln Phe Gln Asn Glu 385
390 395 400 Lys Leu Ile Val Glu Val Leu Lys Val Gly Val Ser Ile Gly
Ala Lys 405 410 415 Asp Tyr Gly Ser Gly Ile Glu Asn His Asp Val Ile
Arg Gly Glu Val 420 425 430 Ile Ala Glu Ser Ile Gly Lys Leu Met Gly
Ser Ser Glu Glu Ser Asp 435 440 445 Ala Ile Gln Arg Lys Ala Lys Asp
Leu Gly Ala Glu Ala Arg Ser Ala 450 455 460 Val Glu Asn Gly Gly Ser
Ser Tyr Asn Asp Val Gly Arg Leu Met Asp 465 470 475 480 Glu Leu Met
Ala Arg Arg Ser Ser Val Lys Val Gly Glu Asp Ile Ile 485 490 495 Pro
Thr Asn Asp Gly Leu 500 5470PRTSolanum lycopersicum 5Met Ser Pro
Lys Leu His Lys Glu Leu Phe Phe His Ser Leu Tyr Lys 1 5 10 15 Lys
Thr Arg Ser Asn His Thr Met Ala Thr Leu Lys Val Leu Met Phe 20 25
30 Pro Phe Leu Ala Tyr Gly His Ile Ser Pro Tyr Leu Asn Val Ala Lys
35 40 45 Lys Leu Ala Asp Arg Gly Phe Leu Ile Tyr Phe Cys Ser Thr
Pro Ile 50 55 60 Asn Leu Lys Ser Thr Ile Glu Lys Ile Pro Glu Lys
Tyr Ala Asp Ser 65 70 75 80 Ile His Leu Ile Glu Leu His Leu Pro Glu
Leu Pro Gln Leu Pro Pro 85 90 95 His Tyr His Thr Thr Asn Gly Leu
Pro Pro Asn Leu Asn Gln Val Leu 100 105 110 Gln Lys Ala Leu Lys Met
Ser Lys Pro Asn Phe Ser Lys Ile Leu Gln 115 120 125 Asn Leu Lys Pro
Asp Leu Val Ile Tyr Asp Ile Leu Gln Arg Trp Ala 130 135 140 Lys His
Val Ala Asn Glu Gln Asn Ile Pro Ala Val Lys Leu Leu Thr 145 150 155
160 Ser Gly Ala Ala Val Phe Ser Tyr Phe Phe Asn Val Leu Lys Lys Pro
165 170 175 Gly Val Glu Phe Pro Phe Pro Gly Ile Tyr Leu Arg Lys Ile
Glu Gln 180 185 190 Val Arg Leu Ser Glu Met Met Ser Lys Ser Asp Lys
Glu Lys Glu Leu 195 200 205 Glu Asp Asp Asp Asp Asp Asp Asp Leu Leu
Val Asp Gly Asn Met Gln 210 215 220 Ile Met Leu Met Ser Thr Ser Arg
Thr Ile Glu Ala Lys Tyr Ile Asp 225 230 235 240 Phe Cys Thr Ala Leu
Thr Asn Trp Lys Val Val Pro Val Gly Pro Pro 245 250 255 Val Gln Asp
Leu Ile Thr Asn Asp Val Asp Asp Met Glu Leu Ile Asp 260 265 270 Trp
Leu Gly Thr Lys Asp Glu Asn Ser Thr Val Phe Val Ser Phe Gly 275 280
285 Ser Glu Tyr Phe Leu Ser Lys Glu Asp Met Glu Glu Val Ala Phe Ala
290 295 300 Leu Glu Leu Ser Asn Val Asn Phe Ile Trp Val Ala Arg Phe
Pro Lys 305 310 315 320 Gly Glu Glu Arg Asn Leu Glu Asp Ala Leu Pro
Lys Gly Phe Leu Glu 325 330 335 Arg Ile Gly Glu Arg Gly Arg Val Leu
Asp Lys Phe Ala Pro Gln Pro 340 345 350 Arg Ile Leu Asn His Pro Ser
Thr Gly Gly Phe Ile Ser His Cys Gly 355 360 365 Trp Asn Ser Ala Met
Glu Ser Ile Asp Phe Gly Val Pro Ile Ile Ala 370 375 380 Met Pro Met
His Leu Asp Gln Pro Met Asn Ala Arg Leu Ile Val Glu 385 390 395 400
Leu Gly Val Ala Val Glu Ile Val Arg Asp Asp Asp Gly Lys Ile His 405
410 415 Arg Gly Glu Ile Ala Glu Thr Leu Lys Gly Val Ile Thr Gly Lys
Thr 420 425 430 Gly Glu Lys Leu Arg Ala Lys Val Arg Asp Ile Ser Lys
Asn Leu Lys 435 440 445 Thr Ile Arg Asp Glu Glu Met Asp Ala Ala Ala
Glu Glu Leu Ile Gln 450 455 460 Leu Cys Arg Asn Gly Asn 465 470
6464PRTOryza sativa 6Met His Val Val Met Leu Pro Trp Leu Ala Phe
Gly His Ile Leu Pro 1 5 10 15 Phe Ala Glu Phe Ala Lys Arg Val Ala
Arg Gln Gly His Arg Val Thr 20 25 30 Leu Phe Ser Thr Pro Arg Asn
Thr Arg Arg Leu Ile Asp Val Pro Pro 35 40 45 Ser Leu Ala Gly Arg
Ile Arg Val Val Asp Ile Pro Leu Pro Arg Val 50 55 60 Glu His Leu
Pro Glu His Ala Glu Ala Thr Ile Asp Leu Pro Ser Asn 65 70 75 80 Asp
Leu Arg Pro Tyr Leu Arg Arg Ala Tyr Asp Glu Ala Phe Ser Arg 85 90
95 Glu Leu Ser Arg Leu Leu Gln Glu Thr Gly Pro Ser Arg Pro Asp Trp
100 105 110 Val Leu Ala Asp Tyr Ala Ala Tyr Trp Ala Pro Ala Ala Ala
Ser Arg 115 120 125 His Gly Val Pro Cys Ala Phe Leu Ser Leu Phe Gly
Ala Ala Ala Leu 130 135 140 Cys Phe Phe Gly Pro Ala Glu Thr Leu Gln
Gly Arg Gly Pro Tyr Ala 145 150 155 160 Lys Thr Glu Pro Ala His Leu
Thr Ala Val Pro Glu Tyr Val Pro Phe 165 170 175 Pro Thr Thr Val Ala
Phe Arg Gly Asn Glu Ala Arg Glu Leu Phe Lys 180 185 190 Pro Ser Leu
Ile Pro Asp Glu Ser Gly Val Ser Glu Ser Tyr Arg Phe 195 200 205 Ser
Gln Ser Ile Glu Gly Cys Gln Leu Val Ala Val Arg Ser Asn Gln 210 215
220 Glu Phe Glu Pro Glu Trp Leu Glu Leu Leu Gly Glu Leu Tyr Gln Lys
225 230 235 240 Pro Val Ile Pro Ile Gly Met Phe Pro Pro Pro Pro Pro
Gln Asp Val 245 250 255 Ala Gly His Glu Glu Thr Leu Arg Trp Leu Asp
Arg Gln Glu Pro Asn 260 265 270 Ser Val Val Tyr Ala Ala Phe Gly Ser
Glu Val Lys Leu Thr Ala Glu 275 280 285 Gln Leu Gln Arg Ile Ala Leu
Gly Leu Glu Ala Ser Glu Leu Pro Phe 290 295 300 Ile Trp Ala Phe Arg
Ala Pro Pro Asp Ala Gly Asp Gly Asp Gly Leu 305 310 315 320 Pro Gly
Gly Phe Lys Glu Arg Val Asn Gly Arg Gly Val Val Cys Arg 325 330 335
Gly Trp Val Pro Gln Val Lys Phe Leu Ala His Ala Ser Val Gly Gly 340
345 350 Phe Leu Thr His Ala Gly Trp Asn Ser Ile Ala Glu Gly Leu Ala
Asn 355 360 365 Gly Val Arg Leu Val Leu Leu Pro Leu Met Phe Glu Gln
Gly Leu Asn 370 375 380 Ala Arg Gln Leu Ala Glu Lys Lys Val Ala Val
Glu Val Ala Arg Asp 385
390 395 400 Glu Asp Asp Gly Ser Phe Ala Ala Asn Asp Ile Val Asp Ala
Leu Arg 405 410 415 Arg Val Met Val Gly Glu Glu Gly Asp Glu Phe Gly
Val Lys Val Lys 420 425 430 Glu Leu Ala Lys Val Phe Gly Asp Asp Glu
Val Asn Asp Arg Tyr Val 435 440 445 Arg Asp Phe Leu Lys Cys Leu Ser
Glu Tyr Lys Met Gln Arg Gln Gly 450 455 460 7515PRTArabidopsis
lyrata 7Met Asp Asp Lys Lys Glu Glu Val Met His Ile Ala Met Phe Pro
Trp 1 5 10 15 Leu Ala Met Gly His Leu Leu Pro Phe Leu Arg Leu Ser
Lys Leu Leu 20 25 30 Ala Gln Lys Gly His Lys Ile Ser Phe Ile Ser
Thr Pro Arg Asn Ile 35 40 45 Leu Arg Leu Pro Lys Leu Pro Ser Asn
Leu Ser Ser Ser Ile Thr Phe 50 55 60 Val Ser Phe Pro Leu Pro Ser
Ile Ser Gly Leu Pro Pro Ser Ser Glu 65 70 75 80 Ser Ser Met Asp Val
Pro Tyr Asn Lys Gln Gln Ser Leu Lys Ala Ala 85 90 95 Phe Asp Leu
Leu Gln Pro Pro Leu Thr Glu Phe Leu Arg Leu Ser Ser 100 105 110 Pro
Asp Trp Ile Ile Tyr Asp Tyr Ala Ser His Trp Leu Pro Ser Ile 115 120
125 Ala Lys Glu Leu Gly Ile Ser Lys Ala Phe Phe Ser Leu Phe Asn Ala
130 135 140 Ala Thr Leu Cys Phe Met Gly Pro Ser Ser Ser Leu Ile Glu
Glu Ser 145 150 155 160 Arg Ser Thr Pro Glu Asp Phe Thr Val Val Pro
Pro Trp Val Pro Phe 165 170 175 Lys Ser Thr Ile Val Phe Arg Tyr His
Glu Val Ser Arg Tyr Val Glu 180 185 190 Lys Thr Asp Glu Asp Val Thr
Gly Val Ser Asp Ser Val Arg Phe Gly 195 200 205 Tyr Thr Ile Asp Gly
Ser Asp Ala Val Phe Val Arg Ser Cys Pro Glu 210 215 220 Phe Glu Pro
Glu Trp Phe Ser Leu Leu Gln Asp Leu Tyr Arg Lys Pro 225 230 235 240
Val Phe Pro Ile Gly Phe Leu Pro Pro Val Ile Glu Asp Asp Asp Asp 245
250 255 Asp Thr Thr Trp Val Arg Ile Lys Glu Trp Leu Asp Lys Gln Arg
Val 260 265 270 Asn Ser Val Val Tyr Val Ser Leu Gly Thr Glu Ala Ser
Leu Arg Arg 275 280 285 Glu Glu Leu Thr Glu Leu Ala Leu Gly Leu Glu
Lys Ser Glu Thr Pro 290 295 300 Phe Phe Trp Val Leu Arg Asn Glu Pro
Gln Ile Pro Asp Gly Phe Glu 305 310 315 320 Glu Arg Val Lys Gly Arg
Gly Met Val His Val Gly Trp Val Pro Gln 325 330 335 Val Lys Ile Leu
Ser His Glu Ser Val Gly Gly Phe Leu Thr His Cys 340 345 350 Gly Trp
Asn Ser Val Val Glu Gly Ile Gly Phe Gly Lys Val Pro Ile 355 360 365
Phe Leu Pro Val Leu Asn Glu Gln Gly Leu Asn Thr Arg Leu Leu Gln 370
375 380 Gly Lys Gly Leu Gly Val Glu Val Leu Arg Asp Glu Arg Asp Gly
Ser 385 390 395 400 Phe Gly Ser Asp Ser Val Ala Asp Ser Val Arg Leu
Val Met Ile Asp 405 410 415 Asp Ala Gly Glu Glu Ile Arg Glu Lys Val
Lys Leu Met Lys Gly Leu 420 425 430 Phe Gly Asn Met Asp Glu Asn Ile
Arg Tyr Val Asp Glu Leu Val Gly 435 440 445 Phe Met Arg Asn Asp Glu
Ser Ser Gln Leu Lys Glu Glu Glu Glu Glu 450 455 460 Asp Asp Cys Ser
Asp Asp Gln Ser Ser Glu Val Ser Ser Glu Thr Asp 465 470 475 480 Glu
Lys Glu Leu Asn Leu Asp Leu Lys Glu Glu Lys Arg Arg Ile Ser 485 490
495 Val Tyr Lys Ser Leu Ser Ser Glu Phe Asp Asp Tyr Val Ala Asn Glu
500 505 510 Lys Met Gly 515 8772PRTOryza sativa 8Met His Val Val
Ile Cys Pro Leu Leu Ala Phe Gly His Leu Leu Pro 1 5 10 15 Cys Leu
Asp Leu Ala Gln Arg Leu Ala Cys Gly His Arg Val Ser Phe 20 25 30
Val Ser Thr Pro Arg Asn Ile Ser Arg Leu Pro Pro Val Arg Pro Ser 35
40 45 Leu Ala Pro Leu Val Ser Phe Val Ala Leu Pro Leu Pro Arg Val
Glu 50 55 60 Gly Leu Pro Asn Gly Ala Glu Ser Thr His Asn Val Pro
His Asp Arg 65 70 75 80 Pro Asp Met Val Glu Leu His Leu Arg Ala Phe
Asp Gly Leu Ala Ala 85 90 95 Pro Phe Ser Glu Phe Leu Gly Thr Ala
Cys Ala Asp Trp Val Met Pro 100 105 110 Thr Ser Ser Ala Pro Arg Gln
Thr Leu Ser Ser Asn Ile His Arg Asn 115 120 125 Ser Ser Arg Pro Gly
Thr Pro Ala Pro Ser Gly Arg Leu Leu Cys Pro 130 135 140 Ile Thr Pro
His Ser Asn Thr Leu Glu Arg Ala Ala Glu Lys Leu Val 145 150 155 160
Arg Ser Ser Arg Gln Asn Ala Arg Ala Arg Ser Leu Leu Ala Phe Thr 165
170 175 Ser Pro Pro Leu Pro Tyr Arg Asp Val Phe Arg Ser Leu Leu Gly
Leu 180 185 190 Gln Met Gly Arg Lys Gln Leu Asn Ile Ala His Glu Thr
Asn Gly Arg 195 200 205 Arg Thr Gly Thr Leu Pro Leu Asn Leu Cys Arg
Trp Met Trp Lys Gln 210 215 220 Arg Arg Cys Gly Lys Leu Arg Pro Ser
Asp Val Glu Phe Asn Thr Ser 225 230 235 240 Arg Ser Asn Glu Ala Ile
Ser Pro Ile Gly Ala Ser Leu Val Asn Leu 245 250 255 Gln Ser Ile Gln
Ser Pro Asn Pro Arg Ala Val Leu Pro Ile Ala Ser 260 265 270 Ser Gly
Val Arg Ala Val Phe Ile Gly Arg Ala Arg Thr Ser Thr Pro 275 280 285
Thr Pro Pro His Ala Lys Pro Ala Arg Ser Ala Ala Pro Arg Ala His 290
295 300 Arg Pro Pro Ser Ser Val Met Asp Ser Gly Tyr Ser Ser Ser Tyr
Ala 305 310 315 320 Ala Ala Ala Gly Met His Val Val Ile Cys Pro Trp
Leu Ala Phe Gly 325 330 335 His Leu Leu Pro Cys Leu Asp Leu Ala Gln
Arg Leu Ala Ser Arg Gly 340 345 350 His Arg Val Ser Phe Val Ser Thr
Pro Arg Asn Ile Ser Arg Leu Pro 355 360 365 Pro Val Arg Pro Ala Leu
Ala Pro Leu Val Ala Phe Val Ala Leu Pro 370 375 380 Leu Pro Arg Val
Glu Gly Leu Pro Asp Gly Ala Glu Ser Thr Asn Asp 385 390 395 400 Val
Pro His Asp Arg Pro Asp Met Val Glu Leu His Arg Arg Ala Phe 405 410
415 Asp Gly Leu Ala Ala Pro Phe Ser Glu Phe Leu Gly Thr Ala Cys Ala
420 425 430 Asp Trp Val Ile Val Asp Val Phe His His Trp Ala Ala Ala
Ala Ala 435 440 445 Leu Glu His Lys Val Pro Cys Ala Met Met Leu Leu
Gly Ser Ala His 450 455 460 Met Ile Ala Ser Ile Ala Asp Arg Arg Leu
Glu Arg Ala Glu Thr Glu 465 470 475 480 Ser Pro Ala Ala Ala Gly Gln
Gly Arg Pro Ala Ala Ala Pro Thr Phe 485 490 495 Glu Val Ala Arg Met
Lys Leu Ile Arg Thr Lys Gly Ser Ser Gly Met 500 505 510 Ser Leu Ala
Glu Arg Phe Ser Leu Thr Leu Ser Arg Ser Ser Leu Val 515 520 525 Val
Gly Arg Ser Cys Val Glu Phe Glu Pro Glu Thr Val Pro Leu Leu 530 535
540 Ser Thr Leu Arg Gly Lys Pro Ile Thr Phe Leu Gly Leu Met Pro Pro
545 550 555 560 Leu His Glu Gly Arg Arg Glu Asp Gly Glu Asp Ala Thr
Val Arg Trp 565 570 575 Leu Asp Ala Gln Pro Ala Lys Ser Val Val Tyr
Val Ala Leu Gly Ser 580 585 590 Glu Val Pro Leu Gly Val Glu Lys Val
His Glu Leu Ala Leu Gly Leu 595 600 605 Glu Leu Ala Gly Thr Arg Phe
Leu Trp Ala Leu Arg Lys Pro Thr Gly 610 615 620 Val Ser Asp Ala Asp
Leu Leu Pro Ala Gly Phe Glu Glu Arg Thr Arg 625 630 635 640 Gly Arg
Gly Val Val Ala Thr Arg Trp Val Pro Gln Met Ser Ile Leu 645 650 655
Ala His Ala Ala Val Gly Ala Phe Leu Thr His Cys Gly Trp Asn Ser 660
665 670 Thr Ile Glu Gly Leu Met Phe Gly His Pro Leu Ile Met Leu Pro
Ile 675 680 685 Phe Gly Asp Gln Gly Pro Asn Ala Arg Leu Ile Glu Ala
Lys Asn Ala 690 695 700 Gly Leu Gln Val Ala Arg Asn Asp Gly Asp Gly
Ser Phe Asp Arg Glu 705 710 715 720 Gly Val Ala Ala Ala Ile Arg Ala
Val Ala Val Glu Glu Glu Ser Ser 725 730 735 Lys Val Phe Gln Ala Lys
Ala Lys Lys Leu Gln Glu Ile Val Ala Asp 740 745 750 Met Ala Cys His
Glu Arg Tyr Ile Asp Gly Phe Ile Gln Gln Leu Arg 755 760 765 Ser Tyr
Lys Asp 770 9442PRTSolanum lycopersicum 9Met Ala Thr Asn Leu Arg
Val Leu Met Phe Pro Trp Leu Ala Tyr Gly 1 5 10 15 His Ile Ser Pro
Phe Leu Asn Ile Ala Lys Gln Leu Ala Asp Arg Gly 20 25 30 Phe Leu
Ile Tyr Leu Cys Ser Thr Arg Ile Asn Leu Glu Ser Ile Ile 35 40 45
Lys Lys Ile Pro Glu Lys Tyr Ala Asp Ser Ile His Leu Ile Glu Leu 50
55 60 Gln Leu Pro Glu Leu Pro Glu Leu Pro Pro His Tyr His Thr Thr
Asn 65 70 75 80 Gly Leu Pro Pro His Leu Asn Pro Thr Leu His Lys Ala
Leu Lys Met 85 90 95 Ser Lys Pro Asn Phe Ser Arg Ile Leu Gln Asn
Leu Lys Pro Asp Leu 100 105 110 Leu Ile Tyr Asp Val Leu Gln Pro Trp
Ala Glu His Val Ala Asn Glu 115 120 125 Gln Asn Ile Pro Ala Gly Lys
Leu Leu Thr Ser Cys Ala Ala Val Phe 130 135 140 Ser Tyr Phe Phe Ser
Phe Arg Lys Asn Pro Gly Val Glu Phe Pro Phe 145 150 155 160 Pro Ala
Ile His Leu Pro Glu Val Glu Lys Val Lys Ile Arg Glu Ile 165 170 175
Leu Ala Lys Glu Pro Glu Glu Gly Gly Arg Leu Asp Glu Gly Asn Lys 180
185 190 Gln Met Met Leu Met Cys Thr Ser Arg Thr Ile Glu Ala Lys Tyr
Ile 195 200 205 Asp Tyr Cys Thr Glu Leu Cys Asn Trp Lys Val Val Pro
Val Gly Pro 210 215 220 Pro Phe Gln Asp Leu Ile Thr Asn Asp Ala Asp
Asn Lys Glu Leu Ile 225 230 235 240 Asp Trp Leu Gly Thr Lys His Glu
Asn Ser Thr Val Phe Val Ser Phe 245 250 255 Gly Ser Glu Tyr Phe Leu
Ser Lys Glu Asp Met Glu Glu Val Ala Phe 260 265 270 Ala Leu Glu Leu
Ser Asn Val Asn Phe Ile Trp Val Ala Arg Phe Pro 275 280 285 Lys Gly
Glu Glu Arg Asn Leu Glu Asp Ala Leu Pro Lys Gly Phe Leu 290 295 300
Glu Arg Ile Gly Glu Arg Gly Arg Val Leu Asp Lys Phe Ala Pro Gln 305
310 315 320 Pro Arg Ile Leu Asn His Pro Ser Thr Gly Gly Phe Ile Ser
His Cys 325 330 335 Gly Trp Asn Ser Ala Met Glu Ser Ile Asp Phe Gly
Val Pro Ile Ile 340 345 350 Ala Met Pro Ile His Asn Asp Gln Pro Ile
Asn Ala Lys Leu Met Val 355 360 365 Glu Leu Gly Val Ala Val Glu Ile
Val Arg Asp Asp Asp Gly Lys Ile 370 375 380 His Arg Gly Glu Ile Ala
Glu Thr Leu Lys Ser Val Val Thr Gly Glu 385 390 395 400 Thr Gly Glu
Ile Leu Arg Ala Lys Val Arg Glu Ile Ser Lys Asn Leu 405 410 415 Lys
Ser Ile Arg Asp Glu Glu Met Asp Ala Val Ala Glu Glu Leu Ile 420 425
430 Gln Leu Cys Arg Asn Ser Asn Lys Ser Lys 435 440
10454PRTEscherichia coli 10Met Gly Thr Glu Val Thr Val His Lys Asn
Thr Leu Arg Val Leu Met 1 5 10 15 Phe Pro Trp Leu Ala Tyr Gly His
Ile Ser Pro Phe Leu Asn Val Ala 20 25 30 Lys Lys Leu Val Asp Arg
Gly Phe Leu Ile Tyr Leu Cys Ser Thr Ala 35 40 45 Ile Asn Leu Lys
Ser Thr Ile Lys Lys Ile Pro Glu Lys Tyr Ser Asp 50 55 60 Ser Ile
Gln Leu Ile Glu Leu His Leu Pro Glu Leu Pro Glu Leu Pro 65 70 75 80
Pro His Tyr His Thr Thr Asn Gly Leu Pro Pro His Leu Asn His Thr 85
90 95 Leu Gln Lys Ala Leu Lys Met Ser Lys Pro Asn Phe Ser Lys Ile
Leu 100 105 110 Gln Asn Leu Lys Pro Asp Leu Val Ile Tyr Asp Leu Leu
Gln Gln Trp 115 120 125 Ala Glu Gly Val Ala Asn Glu Gln Asn Ile Pro
Ala Val Lys Leu Leu 130 135 140 Thr Ser Gly Ala Ala Val Leu Ser Tyr
Phe Phe Asn Leu Val Lys Lys 145 150 155 160 Pro Gly Val Glu Phe Pro
Phe Pro Ala Ile Tyr Leu Arg Lys Asn Glu 165 170 175 Leu Glu Lys Met
Ser Glu Leu Leu Ala Gln Ser Ala Lys Asp Lys Glu 180 185 190 Pro Asp
Gly Val Asp Pro Phe Ala Asp Gly Asn Met Gln Val Met Leu 195 200 205
Met Ser Thr Ser Arg Ile Ile Glu Ala Lys Tyr Ile Asp Tyr Phe Ser 210
215 220 Gly Leu Ser Asn Trp Lys Val Val Pro Val Gly Pro Pro Val Gln
Asp 225 230 235 240 Pro Ile Ala Asp Asp Ala Asp Glu Met Glu Leu Ile
Asp Trp Leu Gly 245 250 255 Lys Lys Asp Glu Asn Ser Thr Val Phe Val
Ser Phe Gly Ser Glu Tyr 260 265 270 Phe Leu Ser Lys Glu Asp Arg Glu
Glu Ile Ala Phe Gly Leu Glu Leu 275 280 285 Ser Asn Val Asn Phe Ile
Trp Val Ala Arg Phe Pro Lys Gly Glu Glu 290 295 300 Gln Asn Leu Glu
Asp Ala Leu Pro Lys Gly Phe Leu Glu Arg Ile Gly 305 310 315 320 Asp
Arg Gly Arg Val Leu Asp Lys Phe Ala Pro Gln Pro Arg Ile Leu 325 330
335 Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys Gly Trp Asn Ser
340 345 350 Val Met Glu Ser Val Asp Phe Gly Val Pro Ile Ile Ala Met
Pro Ile 355 360 365 His Leu Asp Gln Pro Met Asn Ala Arg Leu Ile Val
Glu Leu Gly Val 370 375 380 Ala Val Glu Ile Val Arg Asp Asp Tyr Gly
Lys Ile His Arg Glu Glu 385 390 395 400 Ile Ala Glu Ile Leu Lys Asp
Val Ile Ala Gly Lys Ser Gly Glu Asn 405 410 415 Leu Lys Ala Lys Met
Arg Asp Ile Ser Lys Asn Leu Lys Ser Ile Arg 420 425 430 Asp Glu Glu
Met Asp Thr Ala Ala Glu Glu Leu Ile Gln Leu Cys Lys 435 440 445 Asn
Ser Pro Lys Leu Lys 450 11458PRTStevia rebaudiana 11Met Glu Asn Lys
Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile 1 5 10 15 Leu Phe
Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu 20 25 30
Ala Asn Val Leu Tyr
Ser Lys Gly Phe Ser Ile Thr Ile Phe His Thr 35 40 45 Asn Phe Asn
Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg 50 55 60 Phe
Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro 65 70
75 80 Thr His Gly Pro Leu Ala Gly Met Arg Ile Pro Ile Ile Asn Glu
His 85 90 95 Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met
Leu Ala Ser 100 105 110 Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr
Asp Ala Leu Trp Tyr 115 120 125 Phe Ala Gln Ser Val Ala Asp Ser Leu
Asn Leu Arg Arg Leu Val Leu 130 135 140 Met Thr Ser Ser Leu Phe Asn
Phe His Ala His Val Ser Leu Pro Gln 145 150 155 160 Phe Asp Glu Leu
Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu 165 170 175 Glu Gln
Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser 180 185 190
Ala Tyr Ser Asn Trp Gln Ile Leu Lys Glu Ile Leu Gly Lys Met Ile 195
200 205 Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys
Glu 210 215 220 Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile
Pro Ala Pro 225 230 235 240 Ser Phe Leu Ile Pro Leu Pro Lys His Leu
Thr Ala Ser Ser Ser Ser 245 250 255 Leu Leu Asp His Asp Arg Thr Val
Phe Gln Trp Leu Asp Gln Gln Pro 260 265 270 Pro Ser Ser Val Leu Tyr
Val Ser Phe Gly Ser Thr Ser Glu Val Asp 275 280 285 Glu Lys Asp Phe
Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Lys Gln 290 295 300 Ser Phe
Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp 305 310 315
320 Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Arg Ile Val
325 330 335 Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Gly Ala Ile
Gly Ala 340 345 350 Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu
Ser Val Cys Glu 355 360 365 Gly Val Pro Met Ile Phe Ser Asp Phe Gly
Leu Asp Gln Pro Leu Asn 370 375 380 Ala Arg Tyr Met Ser Asp Val Leu
Lys Val Gly Val Tyr Leu Glu Asn 385 390 395 400 Gly Trp Glu Arg Gly
Glu Ile Ala Asn Ala Ile Arg Arg Val Met Val 405 410 415 Asp Glu Glu
Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln 420 425 430 Lys
Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu 435 440
445 Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu 450 455 12470PRTSolanum
lycopersicum 12Met Ser Pro Lys Leu His Lys Glu Leu Phe Phe His Ser
Leu Tyr Lys 1 5 10 15 Lys Thr Arg Ser Asn His Thr Met Ala Thr Leu
Lys Val Leu Met Phe 20 25 30 Pro Phe Leu Ala Tyr Gly His Ile Ser
Pro Tyr Leu Asn Val Ala Lys 35 40 45 Lys Leu Ala Asp Arg Gly Phe
Leu Ile Tyr Phe Cys Ser Thr Pro Ile 50 55 60 Asn Leu Lys Ser Thr
Ile Glu Lys Ile Pro Glu Lys Tyr Ala Asp Ser 65 70 75 80 Ile His Leu
Ile Glu Leu His Leu Pro Glu Leu Pro Gln Leu Pro Pro 85 90 95 His
Tyr His Thr Thr Asn Gly Leu Pro Pro Asn Leu Asn Gln Val Leu 100 105
110 Gln Lys Ala Leu Lys Met Ser Lys Pro Asn Phe Ser Lys Ile Leu Gln
115 120 125 Asn Leu Lys Pro Asp Leu Val Ile Tyr Asp Ile Leu Gln Arg
Trp Ala 130 135 140 Lys His Val Ala Asn Glu Gln Asn Ile Pro Ala Val
Lys Leu Leu Thr 145 150 155 160 Ser Gly Ala Ala Val Phe Ser Tyr Phe
Phe Asn Val Leu Lys Lys Pro 165 170 175 Gly Val Glu Phe Pro Phe Pro
Gly Ile Tyr Leu Arg Lys Ile Glu Gln 180 185 190 Val Arg Leu Ser Glu
Met Met Ser Lys Ser Asp Lys Glu Lys Glu Leu 195 200 205 Glu Asp Asp
Asp Asp Asp Asp Asp Leu Leu Val Asp Gly Asn Met Gln 210 215 220 Ile
Met Leu Met Ser Thr Ser Arg Thr Ile Glu Ala Lys Tyr Ile Asp 225 230
235 240 Phe Cys Thr Ala Leu Thr Asn Trp Lys Val Val Pro Val Gly Pro
Pro 245 250 255 Val Gln Asp Leu Ile Thr Asn Asp Val Asp Asp Met Glu
Leu Ile Asp 260 265 270 Trp Leu Gly Thr Lys Asp Glu Asn Ser Thr Val
Phe Val Ser Phe Gly 275 280 285 Ser Glu Tyr Phe Leu Ser Lys Glu Asp
Met Glu Glu Val Ala Phe Ala 290 295 300 Leu Glu Leu Ser Asn Val Asn
Phe Ile Trp Val Ala Arg Phe Pro Lys 305 310 315 320 Gly Glu Glu Arg
Asn Leu Glu Asp Ala Leu Pro Lys Gly Phe Leu Glu 325 330 335 Arg Ile
Gly Glu Arg Gly Arg Val Leu Asp Lys Phe Ala Pro Gln Pro 340 345 350
Arg Ile Leu Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys Gly 355
360 365 Trp Asn Ser Ala Met Glu Ser Ile Asp Phe Gly Val Pro Ile Ile
Ala 370 375 380 Met Pro Met His Leu Asp Gln Pro Met Asn Ala Arg Leu
Ile Val Glu 385 390 395 400 Leu Gly Val Ala Val Glu Ile Val Arg Asp
Asp Asp Gly Lys Ile His 405 410 415 Arg Gly Glu Ile Ala Glu Thr Leu
Lys Gly Val Ile Thr Gly Lys Thr 420 425 430 Gly Glu Lys Leu Arg Ala
Lys Val Arg Asp Ile Ser Lys Asn Leu Lys 435 440 445 Thr Ile Arg Asp
Glu Glu Met Asp Ala Ala Ala Glu Glu Leu Ile Gln 450 455 460 Leu Cys
Arg Asn Gly Asn 465 470 13808PRTArabidopsis thaliana 13Met Ala Asn
Ala Glu Arg Met Ile Thr Arg Val His Ser Gln Arg Glu 1 5 10 15 Arg
Leu Asn Glu Thr Leu Val Ser Glu Arg Asn Glu Val Leu Ala Leu 20 25
30 Leu Ser Arg Val Glu Ala Lys Gly Lys Gly Ile Leu Gln Gln Asn Gln
35 40 45 Ile Ile Ala Glu Phe Glu Ala Leu Pro Glu Gln Thr Arg Lys
Lys Leu 50 55 60 Glu Gly Gly Pro Phe Phe Asp Leu Leu Lys Ser Thr
Gln Glu Ala Ile 65 70 75 80 Val Leu Pro Pro Trp Val Ala Leu Ala Val
Arg Pro Arg Pro Gly Val 85 90 95 Trp Glu Tyr Leu Arg Val Asn Leu
His Ala Leu Val Val Glu Glu Leu 100 105 110 Gln Pro Ala Glu Phe Leu
His Phe Lys Glu Glu Leu Val Asp Gly Val 115 120 125 Lys Asn Gly Asn
Phe Thr Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala 130 135 140 Ser Ile
Pro Arg Pro Thr Leu His Lys Tyr Ile Gly Asn Gly Val Asp 145 150 155
160 Phe Leu Asn Arg His Leu Ser Ala Lys Leu Phe His Asp Lys Glu Ser
165 170 175 Leu Leu Pro Leu Leu Lys Phe Leu Arg Leu His Ser His Gln
Gly Lys 180 185 190 Asn Leu Met Leu Ser Glu Lys Ile Gln Asn Leu Asn
Thr Leu Gln His 195 200 205 Thr Leu Arg Lys Ala Glu Glu Tyr Leu Ala
Glu Leu Lys Ser Glu Thr 210 215 220 Leu Tyr Glu Glu Phe Glu Ala Lys
Phe Glu Glu Ile Gly Leu Glu Arg 225 230 235 240 Gly Trp Gly Asp Asn
Ala Glu Arg Val Leu Asp Met Ile Arg Leu Leu 245 250 255 Leu Asp Leu
Leu Glu Ala Pro Asp Pro Cys Thr Leu Glu Thr Phe Leu 260 265 270 Gly
Arg Val Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly 275 280
285 Tyr Phe Ala Gln Asp Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln
290 295 300 Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Ile Glu
Met Leu 305 310 315 320 Gln Arg Ile Lys Gln Gln Gly Leu Asn Ile Lys
Pro Arg Ile Leu Ile 325 330 335 Leu Thr Arg Leu Leu Pro Asp Ala Val
Gly Thr Thr Cys Gly Glu Arg 340 345 350 Leu Glu Arg Val Tyr Asp Ser
Glu Tyr Cys Asp Ile Leu Arg Val Pro 355 360 365 Phe Arg Thr Glu Lys
Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu 370 375 380 Val Trp Pro
Tyr Leu Glu Thr Tyr Thr Glu Asp Ala Ala Val Glu Leu 385 390 395 400
Ser Lys Glu Leu Asn Gly Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser 405
410 415 Asp Gly Asn Leu Val Ala Ser Leu Leu Ala His Lys Leu Gly Val
Thr 420 425 430 Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr
Pro Asp Ser 435 440 445 Asp Ile Tyr Trp Lys Lys Leu Asp Asp Lys Tyr
His Phe Ser Cys Gln 450 455 460 Phe Thr Ala Asp Ile Phe Ala Met Asn
His Thr Asp Phe Ile Ile Thr 465 470 475 480 Ser Thr Phe Gln Glu Ile
Ala Gly Ser Lys Glu Thr Val Gly Gln Tyr 485 490 495 Glu Ser His Thr
Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His 500 505 510 Gly Ile
Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala 515 520 525
Asp Met Ser Ile Tyr Phe Pro Tyr Thr Glu Glu Lys Arg Arg Leu Thr 530
535 540 Lys Phe His Ser Glu Ile Glu Glu Leu Leu Tyr Ser Asp Val Glu
Asn 545 550 555 560 Lys Glu His Leu Cys Val Leu Lys Asp Lys Lys Lys
Pro Ile Leu Phe 565 570 575 Thr Met Ala Arg Leu Asp Arg Val Lys Asn
Leu Ser Gly Leu Val Glu 580 585 590 Trp Tyr Gly Lys Asn Thr Arg Leu
Arg Glu Leu Ala Asn Leu Val Val 595 600 605 Val Gly Gly Asp Arg Arg
Lys Glu Ser Lys Asp Asn Glu Glu Lys Ala 610 615 620 Glu Met Lys Lys
Met Tyr Asp Leu Ile Glu Glu Tyr Lys Leu Asn Gly 625 630 635 640 Gln
Phe Arg Trp Ile Ser Ser Gln Met Asp Arg Val Arg Asn Gly Glu 645 650
655 Leu Tyr Arg Tyr Ile Cys Asp Thr Lys Gly Ala Phe Val Gln Pro Ala
660 665 670 Leu Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr
Cys Gly 675 680 685 Leu Pro Thr Phe Ala Thr Cys Lys Gly Gly Pro Ala
Glu Ile Ile Val 690 695 700 His Gly Lys Ser Gly Phe His Ile Asp Pro
Tyr His Gly Asp Gln Ala 705 710 715 720 Ala Asp Thr Leu Ala Asp Phe
Phe Thr Lys Cys Lys Glu Asp Pro Ser 725 730 735 His Trp Asp Glu Ile
Ser Lys Gly Gly Leu Gln Arg Ile Glu Glu Lys 740 745 750 Tyr Thr Trp
Gln Ile Tyr Ser Gln Arg Leu Leu Thr Leu Thr Gly Val 755 760 765 Tyr
Gly Phe Trp Lys His Val Ser Asn Leu Asp Arg Leu Glu Ala Arg 770 775
780 Arg Tyr Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Pro Leu Ala Gln
785 790 795 800 Ala Val Pro Leu Ala Gln Asp Asp 805
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